Antigens and vaccines directed against human enteroviruses

ABSTRACT

The instant invention provides materials and methods for producing immunologically active antigens derived from members of the Picornaviridae virus family. The picornavirus antigens of the invention may be in a form for use as a vaccine administered to a subject in a therapeutic treatment or for the prevention of a picornavirus infection. The picornavirus antigens of the invention may be in the form of an immunogenic composition for use in vaccines which are administered for the prevention of an Enterovirus infection. The instant invention further encompasses immunogenic compositions comprising Human enterovirus A, Human enterovirus B, Human enterovirus C, Human enterovirus D antigens and their use in vaccines for the prevention of an Enterovirus infection.

FIELD OF THE INVENTION

This invention relates to viruses of the Picornaviridae family, and inparticular antigens and vaccines that may be effective in preventing andtreating infections caused by such viruses.

BACKGROUND OF THE INVENTION

Picornaviruses are a diverse family of viruses which cause a number ofcommon illnesses. Of the Picornaviridae family, viruses of the genusEnterovirus, which are all very closely related, are significant for thenumber of diseases they cause.

Viruses of the genus Enterovirus affect millions of people worldwideeach year, and are often found in the respiratory secretions (e.g.,saliva, sputum, or nasal mucus) and stool of an infected person.Enterovirus infects the gut, thus the derivation of their name from theroot “enteric”. Historically, poliomyelitis was the most significantdisease caused by an enterovirus, that is, poliovirus. There are 62non-polio enteroviruses that can cause disease in humans: 23 Coxsackie Aviruses, 6 Coxsackie B viruses, 28 echoviruses, and 5 otherenteroviruses. Polioviruses, as well as Coxsackie viruses andechoviruses, are spread through the fecal-oral route. Infection canresult in a wide variety of symptoms ranging from mild respiratoryillness (common cold), hand, foot and mouth disease, acute hemorrhagicconjunctivitis, aseptic meningitis, myocarditis, severe neonatalsepsis-like disease, and acute flaccid paralysis.

Of the picornaviruses, Enterovirus represents a genus of a large anddiverse group of small RNA viruses characterized by a singlepositive-strand genomic RNA. All enteroviruses contain a genome ofapproximately 7,500 bases and are known to have a high mutation rate dueto low-fidelity replication and frequent recombination. After infectionof the host cell, the genome is translated in a cap-independent mannerinto a single polyprotein, which is subsequently processed byvirus-encoded proteases into the structural capsid proteins and thenonstructural proteins, which are mainly involved in the replication ofthe virus.

The enteroviruses are associated with several human and mammaliandiseases. Serologic studies have distinguished 66 human Enterovirusserotypes on the basis of antibody neutralization tests. Additionalantigenic variants have been defined within several of the serotypes onthe basis of reduced or nonreciprocal cross-neutralization betweenvariant strains. On the basis of their pathogenesis in humans andanimals, enteroviruses were originally classified into four groups,polioviruses, Coxsackie A viruses (CA), Coxsackie B viruses (CB), andechoviruses, but it was quickly realized that there were significantoverlaps in the biological properties of viruses in the differentgroups.

The Enterovirus genus includes the following ten species:

-   -   Bovine enterovirus    -   Human enterovirus A    -   Human enterovirus B    -   Human enterovirus C    -   Human enterovirus D    -   Human rhinovirus A    -   Human rhinovirus B    -   Human rhinovirus C    -   Porcine enterovirus B    -   Simian enterovirus A

Within these ten species are there are various serotypes, for example:

-   -   Enterovirus serotypes HEV71, EV-76, EV-89, EV-90, EV-91, EV-92        and Coxsackievirus A16 are found under the species Human        enterovirus A.    -   Serotypes Coxsackievirus B1 (CV-B1), CV-B2, CV-B3, CV-B4, CV-B5        (incl. swine vesicular disease virus [SVDV]), CV-B6, CV-A9,        echovirus 1 (E-1; incl. E-8), E-2, E-3, E-4, E-5, E-6, E-7, E-9        (including CV-A23), E-11, E-12, E-13, E-14, E-15, E-16, E-17,        E-18, E-19, E-20, E-21, E-24, E-25, E-26, E-27, E-29, E-30,        E-31, E-32, E-33, enterovirus B69 (EV-B69), EV-B73, EV-B74,        EV-B75, EV-B77, EV-B78, EV-B79, EV-B80, EV-B81, EV-B82, EV-B83,        EV-B84, EV-B85, EV-B86, EV-B87, EV-B88, EV-B93, EV-B97, EV-B98,        EV-B100, EV-B101, EV-B106, EV-B107, EV-B110 (from a chimpanzee)        and the simian enterovirus SA5, are found under the species        Human enterovirus B.    -   Serotypes EV-95, EV-96, EV-99, EV-102, EV-104, EV-105, and        EV-109 are found under the species Human enterovirus C.    -   Serotypes EV-68, EV-70, & EV-94 are found under the species        Human enterovirus D.    -   Poliovirus serotypes PV-1, PV-2, and PV-3 are found under the        species Human enterovirus C.

Diseases caused by enterovirus infection include poliomyelitis which isthe most notable disease caused by an enterovirus infection. Nonspecificfebrile illness is, however, the most common presentation of anenterovirus infection.

Enteroviruses are the most common causes of aseptic meningitis inchildren. In the United States, enteroviruses are responsible for 30,000to 50,000 cases of meningitis. Encephalitis is a rare manifestation ofan enterovirus infection; when it occurs, the most frequent Enterovirusfound to be causing the encephalitis is echovirus 9.

Pleurodynia caused by enteroviruses is characterized by severeparoxysmal pain in the chest and abdomen, along with fever, andsometimes nausea, headache, and emesis.

Pericarditis and/or myocarditis are typically caused by enteroviruses.Arrythmias, heart failure, and myocardial infarction have also beenreported.

Acute hemorrhagic conjunctivitis can be caused by enteroviruses.

Hand, foot and mouth disease is a childhood illness most commonly causedby infection by Coxsackie A virus or HEV71.

A 2007 study suggested that acute respiratory or gastrointestinalinfections associated with enteroviruses may be a factor in chronicfatigue syndrome.

All members of the genus Enterovirus, including HEV71, polioviruses andCoxsackievirus A16 have a single stranded positive sense RNA genomewhich has a single open reading frame encoding a polyprotein, P1,consisting of the capsid proteins VP4, VP2, VP3 and VP1 and severalnon-structural proteins including the viral proteases 3C and 3CD whichare responsible for cleaving the polyprotein P1 into individual capsidproteins VP1, VP3 and VP0, which VP0 is eventually cleaved into VP2 andVP4. The capsid proteins may assemble into virus like particles (VLPs).

Human enterovirus 71 (HEV71) and Coxsackievirus A16 are Enterovirusserotypes notable as the major causative agents for hand, foot and mouthdisease (HFMD), and HEV71 is sometimes associated with severe centralnervous system diseases. HEV71 was first isolated and characterized fromcases of neurological disease in California in 1969. To date, little isknown about the molecular mechanisms of host response to HEV71infection, but increases in the level of mRNAs encoding chemokines,proteins involved in protein degradation, complement proteins, andpro-apoptotic proteins have been implicated.

Hand Foot and Mouth Disease (HFMD) is a common, self-limiting illness ofchildren caused by a group of species A enteroviruses (Picornaviridaefamily) such as human Coxsackievirus A16 (CVA16), Coxsackievirus A10(CVA10) and Human enterovirus A 71 (HEV71). The virus is excreted infeces and is also found in pharyngeal secretions. Transmission isassociated with close contact among children and through environmentalcontamination. The disease is characterized by an acute onset of feverwith a rash on the palms, soles, buttocks, and knees, and vesicles onbuccal membranes that usually resolve in 7-10 days. Only a smallproportion of children with HFMD develop severe disease.

Severe disease involving primarily the neurologic and cardiovascularsystems manifesting as syndromes such as meningitis, encephalitis, acuteflaccid paralysis, pulmonary edema and cardiac failure generally occuronly with HEV71 infection. In the Asia-Pacific Region the mostdevastating neurological syndrome is brainstem encephalitis, which has amortality rate of 40-80 percent. Children with severe HFMD may takemonths to recover, and in some cases the neurologic damage may bepermanent. Currently, there is no specific antiviral treatment for HFMDand no vaccines to prevent enterovirus infection other than polio.

HEV71 was first isolated from a child who died of encephalitis inCalifornia in 1969, and first reported in 1974. Although the virus hasbeen detected worldwide since then, the recent regional epidemics ofHFMD in Asia has raised concern that more pathogenic forms of HEV71 maybe emerging in the region. The first recognition of a HFMD outbreak witha high number of fatalities was in Sarawak, Malaysia in 1997. The virusassociated with the outbreak then was HEV71. Taiwan reported 129,106HFMD cases in a 1998 epidemic with 405 having severe disease with 78deaths. Singapore reported an epidemic of 9000 cases with 7 deathsduring 2000-2001, and since then has experienced recurrent epidemicsevery two to three years. During the first 8 months of 2008, Singaporereported 19,530 cases and one death due to HFMD. Since then HEV71outbreaks have been reported regularly in Singapore, Thailand, Malaysia,Taiwan, Japan, Korea and Vietnam.

China reported 83,344 cases with 17 deaths in 2007, and in 2008experienced a large outbreak in Fuyang City in Anhui Province spreadingthroughout many parts of China. These large outbreaks were widelycovered by the press, which highlighted parental concerns about thehealth of their children and the social disruption from closing ofschools and day care centers by public health departments in an attemptto break the chain of transmission. Since then China has reported largeoutbreaks annually.

With regard to disease caused by other members of the Picornaviridaefamily, natural infection and prevalence of polio have occurredexclusively in the human being since ancient times as an infectiousdisease. A large number of humans still become infected with polio everyyear in developing countries. Hence, the eradication of polio is anongoing process.

Polioviruses were formerly classified as a species belonging to thegenus Enterovirus in the family Picornaviridae. The Poliovirus specieshas been eliminated from the genus Enterovirus. The poloviruses areclassified as serotypes, Human poliovirus 1 (PV-1), Human poliovirus 2(PV-2), and Human poliovirus 3 (PV-3), and are considered to be subtypesof species Human enterovirus C, in the genus Enterovirus in the familyPicornaviridae. The type species of the genus Enterovirus was changedfrom Poliovirus to Human enterovirus C in 2008.

The three subtypes of species Human enterovirus C, PV-1, PV-2 and PV-3,are characterized by a slightly different capsid protein. Capsidproteins define cellular receptor specificity and virus antigenicity.PV-1 is the most common form encountered in nature; however, all threeforms are extremely infectious and can affect the spinal cord and causepoliomyelitis.

Infection with Human enterovirus C has been a widespread problem andinactivated whole virus vaccines have been used for mass immunizationand are currently available. Good results have been obtained withinactivated poliomyelitis vaccines which may be prepared according to amethod which has been developed by Salk and has been improved later inseveral aspects. Generally, these vaccines contain a mixture ofinactivated polio virus of strains Mahoney, MEF1 and Saukett.

Although an attenuated Human enterovirus C has been produced and used asan attenuated oral polio vaccine, the attenuated Human enterovirus C maybe dangerous because of the possible reversion of pathogenicity(paralysis-based neurovirulence) in persons administered to, or incontact with, whole viruses. Hence, there is a need for a safe andeffective polio vaccine which is free of such pathogenicity.

Like all enteroviruses, four different Human enterovirus C coat/capsidpolypeptides have been identified and are designated as VP1, VP2, VP3and VP4, which associate to form an icosahedral virus capsid. Typically,vaccination with the individual polypeptides of Human enterovirus C hasshown that the isolated polypeptides are not capable of raisingneutralizing antibodies in humans and animals (Meloen, et al., J. Gen.Virol. 45:761-763, 1979).

U.S. Pat. No. 4,508,708 teaches that individual polypeptides of poliovirus and hand, foot and mouth disease virus, VP1, VP2, VP3 and VP4, arenot capable of raising neutralizing antibodies in humans and animals andthat, among the individual polypeptides of the hand, foot and mouthdisease virus, only VP1 possesses this capability. U.S. Pat. No.4,508,708 demonstrates that, among the Human enterovirus C type 2 MEF1virion VP1, VP2 and VP3 polypeptides, only the VP3 is capable ofinducing neutralizing antibodies, although the antibody titer is low. Itwas found, however, that VP1, VP2 and VP3 are capable of inducingneutralizing antibodies only when the immunization is carried out with apreparation containing arildone, a broad spectrum antiviral agent thathas been shown to selectively inhibit replication of picornaviruses(Langford, et al. Antimicrobial Agents and Chemotherapy 28:578-580,1985).

Thus, the problem to be solved is the preparation of an effectivevaccine which provides protective immunity against a human enterovirusinfection, and without the use of antiviral compounds. The humanenteroviruses for which protection is desired are, for example Humanenterovirus A, including Coxsackievirus A16 and Human enterovirus 71;Human enterovirus B, including Coxsackievirus B serotypes, echovirusesand enterovirus serotypes; Human enterovirus C, including Humanpoliovirus 1, Human poliovirus 2 and Human poliovirus 3; as well asHuman enterovirus D, including EV 68.

For the purposes of the instant invention, a vaccine is understood bythose skilled in the art and may further be defined as a prophylactic ortherapeutic material containing antigens derived from one morepathogenic organisms which, upon administration to a human subject oranimal, will stimulate active immunity and protect against infectionwith these or related organisms (i.e., produce protective immunity).

Furthermore, protective immunity may be well understood by those skilledin the art. Nonetheless, protective immunity comprises, at least, theinduction, or elicitation of neutralizing antibodies and/or T-cellimmune response which will neutralize the virus.

It is well recognized in the vaccine art, that it is unclear whether anantigen derived from a pathogen will elicit protective immunity. Ellis(Chapter 29 of Vaccines, Plotkin, et al. (eds) WB Saunders,Philadelphia, at page 571,1998) exemplifies this problem in therecitation that “the key to the problem (of vaccine development) is theidentification of that protein component of a virus or microbialpathogen that itself can elicit the production of protective antibodies. . . , and thus protect the host against attack by the pathogen.”

An approach to making improved vaccines against picornaviruses would beto mimic the virus capsid structure or its components which may elicitprotective antibodies such as are produced with a killed whole virusvaccine. This kind of approach is safer than a killed, inactivated orattenuated vaccine approach because there is no opportunity forreversion.

All picornaviruses share the same genomic structure, including 4structural genes within the P1 gene: VP1, VP2, VP3, and VP4, the VP4 andVP2 being expressed together as VP0, and viral proteases within the 3Cand 3D genes. The viral protease will cleave the P1 gene, therebyallowing the virus to assemble into virus like particles (VLPs), viruscapsomers, complexes and/or antigens of enteroviruses.

Vaccines have been proposed with indifferent success. It has beenproposed to use subunit vaccines comprising the major capsid protein,VP1, of enteroviruses, as the basis of vaccines for the prevention andtreatment of enterovirus infections, including HEV71 infections (Wu, etal., 2001).

With regard to prophylaxis against enterovirus infection, it is possibleto envisage a killed virus vaccine approach, which has been shown toelicit protective antibodies. The low virus titres achieved in generalmakes manufacturing of such enterovirus vaccines a challenge.

The present invention pertains to vaccines including antigeniccoat/capsid proteins of viruses of the Picornaviridae family and whichvaccines are devoid of virus RNA which may contribute to neurovirulence.The vaccines of the invention may comprise as antigens, the polypeptidesP1 or VP0, or capsid proteins (VP's), designated as VP1, VP2, VP3 and/orVP4, or immunologically or biologically active fragments thereof whichelicit neutralizing antibodies against enteroviruses.

The present invention relates to vaccines in which the picornavirusantigens are present in the form of one or more picornaviruspolypeptides, especially human Enterovirus peptides VP2, VP3 or VP0,immunogenic fragments thereof, and/or antigenic determinants thereof.The picornavirus polypeptides may be obtained by chemical synthesis orby means of recombinant DNA techniques using known human Enterovirusamino acid or nucleic acid sequences.

SUMMARY OF THE INVENTION

A vaccine comprising one or more immunologically active antigenscomprising one or more human Enterovirus polypeptides selected from VP0,VP1, VP2, VP3, VP4, and immunologically active fragments thereof, such a

vaccine, which elicits a protective and/or neutralizing immune responsedirected against a human Enterovirus, such a

vaccine, wherein the human Enterovirus is selected from Humanenterovirus A, Human enterovirus B, Human enterovirus C and Humanenterovirus D, such a

vaccine, wherein the Human enterovirus A is selected from Humanenterovirus 71 (HEV71) and Coxsackievirus A16, wherein the Humanenterovirus B is selected from Coxsackievirus B and echovirus, whereinthe Human enterovirus C is selected from Human poliovirus 1, Humanpoliovirus 2 and Human poliovirus 3, and wherein the Human enterovirus Dis EV68, such a

vaccine, wherein the vaccine comprises an immunologically activeEnterovirus VP0 polypeptide, such a

vaccine, wherein the vaccine comprises an immunologically activeEnterovirus VP2 polypeptide, such a

vaccine, wherein the vaccine comprises an immunologically activeEnterovirus VP3 polypeptide, such a

vaccine, wherein the vaccine comprises an immunologically activeEnterovirus VP1 polypeptide, such a

vaccine, wherein the vaccine comprises polypeptides from one or moreEnterovirus species or serotype, such a

vaccine, wherein the species or serotype may be Human enterovirus Aselected from HEV71 and Coxsackievirus A16, such a

vaccine, wherein the species or serotype may be Human enterovirus Cselected from PV-1, PV-2 and PV-3, such a

vaccine, wherein the one or more immunologically active antigenscomprising one or more human Enterovirus polypeptides selected from VP0,VP1, VP2, VP3, VP4, and immunologically active fragments thereof, are inthe form of a virus-like particle, capsomer, complex and/or aggregate,such a

vaccine, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP0 polypeptide, such a

vaccine, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP2 polypeptide, such a

vaccine, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP3 polypeptide, such a

vaccine, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP1 polypeptide, a

method of vaccinating a subject against an Enterovirus infection,comprising administering to the subject a vaccine comprising one or moreimmunologically active antigens comprising one or more human Enteroviruspolypeptides selected from VP0, VP1, VP2, VP3, VP4, and immunologicallyactive fragments thereof, in an amount effective to elicit a protectiveand/or neutralizing immune response when administered to the subject,such a

method, wherein the vaccine comprises an immunologically activeEnterovirus VP0 polypeptide, such a

method, wherein the vaccine comprises an immunologically activeEnterovirus VP1 polypeptide, such a

method, wherein the vaccine comprises an immunologically activeEnterovirus VP3 polypeptide, such a

method, wherein the vaccine comprises an immunologically activeEnterovirus VP2 polypeptide, such a

method, wherein the one or more immunologically active antigenscomprising one or more human Enterovirus polypeptides selected from VP0,VP1, VP2, VP3, VP4, and immunologically active fragments thereof, are inthe form of a virus-like particle, capsomer, complex and/or aggregate,such a

method, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP0 polypeptide, such a

method, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP2 polypeptide, such a

method, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP3 polypeptide, such a

method, wherein the virus-like particle comprises an immunologicallyactive Enterovirus VP1 polypeptide, such a

method, wherein the vaccine comprises polypeptides from one or moreEnterovirus species or serotype, such a

method, wherein the Enterovirus species or serotype may be Humanenterovirus A selected from HEV71 and Coxsackievirus A16, such a

method, wherein the Enterovirus species or serotype may be Humanenterovirus C selected from PV-1, PV-2 and PV-3, an

expression cassette comprising a promoter operably linked to a nucleicacid encoding a human Enterovirus P1 polypeptide, an Internal RibosomeEntry Site (IRES), and a nucleic acid encoding a human Enterovirus 3CDprotease, such an

expression cassette, wherein the polypeptide is human Enterovirus P1,such an

expression cassette, wherein the human Enterovirus 3CD proteaseprocesses the human Enterovirus P1 polypeptide, such an

expression cassette, wherein the processed human Enterovirus P1polypeptides associate to form virus like particles, capsomers,complexes and/or aggregates, such an

expression cassette, wherein the human Enterovirus P1 polypeptidecomprises combinations of polypeptides selected from VP0, VP1, VP2, VP3,VP4, and immunologically active fragments thereof, such an

expression cassette, wherein the human Enterovirus is selected fromHuman enterovirus A and Human enterovirus C, such an

expression cassette, wherein the Human enterovirus A is selected fromHEV71 and Coxsackievirus A16, such an

expression cassette, wherein the Human enterovirus C is selected fromHuman poliovirus 1 (PV-1), Human poliovirus 2 (PV-2) and Humanpoliovirus 3 (PV-3), such an

expression cassette, wherein the IRES is derived fromEncephalomyocarditis virus (EMCV), such an

expression cassette, wherein the IRES derived from Encephalomyocarditisvirus (EMCV) has been genetically modified to reduce IRES activity, suchan

expression cassette, wherein the IRES derived from EMCV has beengenetically modified by adding one nucleotide (A7) to the A6 bifurcationloop in the JK segment, such an

expression cassette, wherein the IRES is derived from a humanEnterovirus, such an

expression cassette, wherein the promoter is a eukaryotic promoter, suchan

expression cassette, wherein the eukaryotic promoter is a polyhedrinpromoter, such an

expression cassette, wherein the promoter is operably linked to anucleic acid encoding a Human enterovirus A P1 polypeptide, an EMCVIRES, and a Human enterovirus A 3CD protease, such an

expression cassette, wherein the EMCV IRES has been mutated to reduceIRES activity, such an

expression cassette, wherein the promoter is operably linked to anucleic acid encoding a Human enterovirus A P1 polypeptide, an HEV71IRES, and a Human enterovirus A 3CD protease, such an

expression cassette, wherein the promoter is operably linked to anucleic acid encoding a Human enterovirus A P1 polypeptide, a Humanenterovirus C IRES, and a Human enterovirus A 3CD protease, such an

expression cassette, wherein the promoter is operably linked to anucleic acid encoding a Human enterovirus C P1 polypeptide, a Humanenterovirus C IRES, and a Human enterovirus C 3CD protease, such an

expression cassette, wherein the promoter is operably linked to anucleic acid encoding a Human enterovirus C P1 polypeptide, an HEV71IRES, and a Human enterovirus C 3CD protease, such an

expression cassette, wherein the promoter is operably linked to anucleic acid encoding a Human enterovirus C P1 polypeptide, an EMCVIRES, and a Human enterovirus C 3CD protease, such a

method of making a vaccine comprising introducing an VLP expressioncassette into a host cell, culturing the host cell for a period of timesufficient to produce the polypeptides of the expression cassette andrecovering human Enterovirus polypeptides from the host cell and/orculture supernatant, such a

method, wherein the host cell is a eukaryotic cell, such a

method, wherein the eukaryotic cell is selected from insect cells,mammalian cell lines and yeast cells, such a

method, wherein the insect cells are selected from Spodopterafrugiperda, Trichoplusia ni, drosophila, and mosquito cells derived fromAedes albopictus, such a

method, wherein the mammalian cell lines are selected from CHO, HEK 293,COS-1, HeLa, Vero and NIH3T3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HEV71 VLP expression cassette [P1+IRES+3CD] and the pSN01plasmid.

FIG. 2. HEV71 VLP expression cassette [P1+IRES+3C] and the pSN03plasmid.

FIG. 3. Expression of VP1 in supernatant of SN07 infected Sf9 cells.

FIG. 4. Processed VP1 in both the supernatants and the lysates.

FIG. 5. VP1 and VP0 are in the retentate after ultrafiltration over a100 kDa molecular weight cut off (MWCO) membrane.

FIG. 6. Immunization schedules to produce neutralizing antibodies.

FIG. 7. Immunoblots probed with rabbit polyclonal antisera against VP0(arrow) at 3 different times of harvest.

FIG. 8. Pooled neutralizing sera from mice immunized with the oligomericantigens in the supernatant of SN07 infected Sf9 cells have high titresagainst recombinant VP2 in ELISA.

FIG. 9. Pooled neutralizing sera from mice immunized with oligomericantigens in the supernatant of SN07 infected Sf9 cells bind morestrongly to VP2 and VP0 than to VP1.

FIG. 10. EMCV IRES (SEQ ID NO:1) region of the EMCV genome.

FIG. 11. Out framing of the EMCV start codon with 3CD protease codingsequence; native IRES sequence (SEQ ID NO:2) versus mutant IRES sequence(SEQ ID NO:3).

FIG. 12. Plasmid pSN01-M1.

FIG. 13. Plasmid pSN01-M2

FIG. 14. Plasmid pSN01-M3.

FIG. 15. Comparison of expression of different recombinant baculovirusdesigns for expression of HEV71 VLP's.

FIG. 16. Plasmid pFastBac™ HT.

FIG. 17. Prokaryotic expression construct for antigenic fusion proteinsof Human enterovirus A and Human enterovirus C.

FIG. 18. Antibodies from pooled neutralizing sera from mice immunizedwith SN07 retentate binds to all the components of HEV71 VLPs.

FIG. 19. Characterization of HEV71 VLPs, pull-down of HEV71 VLPs fromthe culture supernatant.

FIG. 20. Analysis of affinity column (AFC) purified HEV71 VLPs.

FIG. 21. An electron micrograph picture of AFC purified HEV71 VLPs.

FIG. 22. Human enterovirus C (poliovirus-PV) VLP expression.

FIG. 23. Poliovirus-VLP VP3-VP1 ELISA.

FIG. 24. HEV71 VLP expression cassette with HEV71-IRES and HEV71 3CDprotease.

FIG. 25. HEV71 VLP expression cassette with PV-IRES and HEV71 3CDprotease.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides virus like particles (VLPs), virus capsomers,aggregates, and complexes of antigens from viruses of the Picornaviridaefamily as an immunogenic composition and/or vaccine for the protectionagainst and/or treatment of a picornavirus infection. Representativeexamples may include an Enterovirus, a Coxsackie virus, and apoliovirus.

The invention in another aspect provides virus proteins for example, aP1 protein or a combination of picornavirus VP0 proteins, VP1 proteins,VP2 proteins, VP3 proteins, and/or VP4 proteins, or immunologically orbiologically active fragments thereof, which elicit neutralizingantibodies. The invention includes fusion-proteins comprising theaforementioned virus proteins and/or fragments thereof, which fusionproteins are immunologically active or biologically active to elicitproduction of neutralizing antibodies which are protective.

In an embodiment, an Enterovirus antigen may be a combination ofEnterovirus coat/capsid proteins, or immunologically active fragmentsthereof. The virus coat/capsid proteins may be any combination of VP0,VP1, VP2, VP3 and/or VP4 proteins, and may take the form of a virus-likeparticle (VLP), capsomer, complex and/or aggregate. The combination maybe in the form of a fusion protein.

The invention in an additional aspect includes a method for productionof Picornaviridae virus like particles (VLPs), capsomers, complexesand/or aggregates which may include the steps of: (i) constructing anexpression cassette operably linked to a promoter comprising one or morenucleic acids which each encode a picornavirus protein, for example, aP1 protein or a combination of picornavirus VP0 protein, VP1 protein,VP2 protein, VP3 protein, and/or a VP4 protein, which is/are operablylinked to an internal ribosome entry site (IRES), which IRES is operablylinked to a 3C or 3CD protease; (ii) transfecting or transforming asuitable host cell with the expression cassette; (iii) culturing thehost cells under conditions in which virus like particles (VLPs) and/orcapsomers and/or antigens are produced by the cell after expression ofthe nucleic acids comprised in the cassette.

A nucleic acid or recombinant DNA molecule may be obtained whereby openreading frames which encode Coxsackievirus A16, HEV71, Human enterovirusC (human polioviruses PV1, PV2 and/or PV3), EV 68, or any otherpicornavirus proteins and proteases may be amplified by PCRamplification using suitably designed primers complementary to nucleicacid sequences of Coxsackievirus A16, HEV71 or Human enterovirus C orany other picornavirus. Suitable primers may be designed according tostandard techniques from publicly available nucleic acid sequences ofenteroviruses, including Coxsackievirus A16, HEV71 and Human enterovirusC or any other picornavirus. Complete genome sequences are available inGenBank and are accessible at the National Center for BiotechnologyInformation (NCBI).

In an embodiment, a picornavirus P1 protein, or any Enterovirus P1protein, is expressed as a polypeptide which is subsequently cleaved bythe 3C or 3CD protease into VP0, VP1 and VP3 virus protein, orimmunologically or biologically active fragments thereof, whichEnterovirus proteins elicit neutralizing antibodies directed againstenteroviruses. The VP0 protein may be further cleaved into VP2 and VP4proteins, or immunologically or biologically active fragments thereofwhich elicit neutralizing antibodies directed against enteroviruses. Thevirus proteins may self-assemble into VLPs, capsomers and/or aggregatesof enterovirus proteins. Further it will be appreciated that theprotease genes may be included in the same DNA recombinant molecule ofthe VLP expression cassette or in different DNA recombinant molecules,and/or expressed from different promoters or translation elements.

Recombinant DNA molecules and nucleic acids of the VLP expressioncassettes may be devised whereby open reading frames which encodepicornavirus structural proteins or proteases may be obtained by PCRamplification using suitably designed primers complementary to nucleicacid sequences of human picornaviruses.

In a further embodiment, the recombinant DNA molecule may encode afusion protein having at least two enterovirus structural proteins, orportions thereof, which are expressed as a single polypeptide antigen.

The present invention encompasses a VLP expression cassette whichharbors the gene sequences for Enterovirus structural proteins (P1region) with a protease (3CD) which is necessary for the processing ofP1 proteins into the proteins of the virus capsid, thus allowing theself-assembly of Enterovirus VLPs. The expression cassette is abicistronic vector which uses a promoter upstream of the nucleic acidcoding sequence for an Enterovirus P1 protein. Downstream from thecistron encoding the P1 protein is an internal ribosome entry site(IRES) sequence followed by the cistron containing a nucleotide sequenceencoding the 3CD protease.

Expression of the P1 region and the 3CD protease proceeds from a singlebicistronic message wherein the 3CD protease gene is translated in acap-independent fashion under the control of the IRES. It is observedthat expression of the protease 3CD may be moderately toxic leading topremature death of the host cells, thereby lowering the yield of theEnterovirus capsid proteins and VLPs. The activity of the protease maybe reduced while maintaining the high level of P1 protein expressionfrom the cassette. Different IRESs and IRES sequences comprisingmutations were inserted into the expression cassettes to controlexpression/activity of the 3CD protease and to identify effective IRESto properly process the P1 without being toxic to the cell. Forefficient production of VLPs, a number of recombinant baculoviruseswhich have the complete P1 coding sequence and the complete 3CD proteasecoding sequence whose expression is under the control of IRESs fromdifferent species or serotypes of viruses, were tested for efficientproduction of VLPs.

For example, the expression cassette of invention may comprise apromoter which is operably linked to a nucleic acid encoding a Humanenterovirus A P1 polypeptide, an EMCV IRES, and a Human enterovirus A3CD protease.

The expression cassette of invention may comprise a promoter which isoperably linked to a nucleic acid encoding a Human enterovirus A P1polypeptide, a Human enterovirus C IRES, and a Human enterovirus A 3CDprotease.

The expression cassette of invention may comprise a promoter which isoperably linked to a nucleic acid encoding a Human enterovirus C P1polypeptide, an HEV71 IRES, and a Human enterovirus C 3CD protease.

Furthermore, the expression cassette of invention may comprise apromoter which is operably linked to a nucleic acid encoding a Humanenterovirus C P1 polypeptide, an EMCV IRES, and a Human enterovirus C3CD protease.

Moreover, making truncations and mutations of the 3CD protease in theexpression cassette which comprises efficient IRES may achieve maximumyield of VLPs. For example, the Glycine of the HEV71 3C protease whichis amino acid 1671 of GenBank accession number DQ341362.1 is changed toan Alanine using site directed mutagenesis for the expression of mutantHEV71 3C and subsequent processing of an HEV71 P1 polypeptide.

Counter to conventional wisdom in the art with respect to the goal ofachieving high levels of expression and activity of a protein from anexpression cassette, in an embodiment, the instant invention actuallyseeks to reduce the activity of a protein to achieve a maximum proteinyield. Mutation of the IRES or 3C protease nucleic acid to reduceactivity unexpectedly results in an increased yield of Enteroviruscapsid proteins and VLPs.

The expression cassettes may be cloned into suitable vectors andtransformed/transfected into appropriate host cells for expression andpurification of antigens for vaccines and protection against infectionsfrom picornaviruses, including enteroviruses.

The expression cassettes encoding picornavirus antigens may be comprisedin plasmids which may be transfected into eukaryotic host cells andexpressed under the appropriate growth conditions. Suitable eukaryoticexpression systems are known to those skilled in the art and includeinducible expression systems and appropriate eukaryotic host cells.

Mammalian cell expression vectors comprising an expression cassette ofthe invention include those which may be transiently transfected intohost cells and cell lines. Moreover, mammalian cell expression vectorsmay be vectors which are stably maintained within the host cellfollowing transfection.

Furthermore, mammalian cell expression vectors may include vectors whichare stably or transiently transfected into mammalian host cells or celllines wherein expression of the protein of interest is induced by theaddition of an inducing agent into the culture medium. Mammalian hostcells and cell lines include, for example, CHO, HEK 293, COS-1, HeLa,Vero and NIH3T3 cells. It will also be appreciated that other eukaryotichost cells may include yeast cells or other mammalian cell lines.

The expression cassette may be contained in recombinant viruses whichmay transfect the host cell. Suitable viruses that may be used for thispurpose include baculovirus, vaccinia, sindbis virus, SV40, Sendaivirus, retrovirus and adenovirus. Suitable host cells may include hostcells that are compatible with the above viruses and these includeinsect cells such as Spodoptera frugiperda (e.g. Sf9 cells) Trichoplusiani, CHO cells, chicken embryo fibroblasts, BHK cells, human SW13 cells,drosophila, mosquito cells derived from Aedes albopictus.

The expression cassette comprising Enterovirus nucleic acids may beintroduced into an appropriate host cell by means known to those skilledin the art. The host cells are propagated and cultured under conditionswhich allow expression of Enterovirus genes and proteins.

A gene encoding an enterovirus VP2 protein, or immunologically orbiologically active fragments thereof which elicit neutralizingantibodies against enteroviruses, may be inserted in a plasmidcontaining a suitable promoter and expressed in a host cell. Theproduced enterovirus VP2 protein will be isolated and used as the basisof an immunogenic composition for use as a vaccine or for diagnosticuse.

A gene encoding an enterovirus VP4 protein, or immunologically orbiologically active fragments thereof which elicit neutralizingantibodies against enteroviruses, may be inserted in a plasmidcontaining a suitable promoter and expressed in a host cell. Theproduced enterovirus VP4protein will be isolated and used as the basisof an immunogenic composition for use as a vaccine or for diagnosticuse.

A gene encoding an enterovirus VP0 protein, or immunologically orbiologically active fragments thereof which elicit neutralizingantibodies against enteroviruses, may be inserted in a plasmidcontaining a suitable promoter and expressed in a host cell. Theproduced enterovirus VP0 protein will be isolated and used as the basisof an immunogenic composition for use as a vaccine or for diagnosticuse.

A gene encoding an enterovirus VP0 protein may be operably linked to asuitable promoter and inserted into a plasmid, which plasmid exhibits anenterovirus protease linked to a suitable promoter to provide a doublyrecombinant plasmid, which doubly recombinant plasmid may ultimately beexpressed in a eukaryotic or prokaryotic cell expression system.

Suitable vectors for the cloning of genes and expression of enteroviruspolypeptide antigens include cosmids or plasmids. Suitable expressionsystems include prokaryotic expression systems known to those skilled inthe art and prokaryotic host cells, including E. coli, transformed withthe cosmids or plasmids for expression of proteins in prokaryotic cells.

Suitable expression systems include eukaryotic expression systems knownto those skilled in the art and eukaryotic host cells transformed withplasmids for expression of proteins in various eukaryotic host cells andcell lines.

Moreover, the Enterovirus polypeptide antigens may be obtained from hostcells or culture supernatants by means known to those skilled in theart.

The Enterovirus VLPs, capsomers, antigens, immunologically activecomponents thereof, and/or aggregates thereof, may be obtained fromtransfected and/or transformed host cells, or host cell culture medium,supernatants and lysates by any suitable means of purification known tothose skilled in the art. Isolation of proteins released into theculture medium is a facile method of obtaining Enterovirus VLPs,capsomers, antigens and/or aggregates. The Enterovirus VLPs, capsomers,antigens, immunologically active components thereof, and/or aggregatesthereof, may be further concentrated and purified by means known tothose skilled in the art.

The invention in another aspect includes a vaccine containingpicornavirus antigens, such as Enterovirus antigens, VLPs and/orcapsomers in combination with a suitable adjuvant. The picornavirusantigens, immunologically active fragments thereof, VLPs and/orcapsomers may be combined with any suitable adjuvant such as ModifiedVaccinia Virus, ISCOMS, alum, aluminum hydroxide, aluminum phosphate,Freund's Incomplete or Complete Adjuvant, Quil A and other saponins orany other adjuvant as described for example in Vanselow (1987) S. Vet.Bull. 57 881-896.

The meaning of the terms “aluminum phosphate” and “aluminum hydroxide”as used herein includes all forms of aluminum hydroxide or aluminumphosphate which are suitable for adjuvanting vaccines.

Moreover, picornavirus antigens may be prepared by chemical synthesis ofpolypeptides based on the publicly available nucleic acid or proteinsequences of human poliovirus or by chemical synthesis.

In an aspect, the invention provides a vaccine comprising one or moreHuman enterovirus C antigen(s). As used herein the expression“poliovirus antigen” or “Human enterovirus C antigen” refers to anyantigen capable of stimulating neutralizing antibodies to Humanenterovirus C. The virus antigen may comprise a coat/capsid protein, orfragments thereof, antigenic determinants, and/or other Humanenterovirus C proteins.

The invention in one aspect includes Human enterovirus C virus subunitvaccines comprising a single Human enterovirus C virus coat/capsidprotein as an antigen. More specifically the invention pertains tovaccines comprising a Human enterovirus C VP2 coat/capsid protein, orimmunogenic fragment thereof, as antigen.

The invention in another aspect includes a vaccine comprising Humanenterovirus C virus like particles (VLPs), capsomers, complexes and/oraggregates, comprising Human enterovirus C VP1, VP2, VP3 and/or VP4, orVP0 proteins.

A recombinant DNA molecule may be obtained whereby nucleic acidscomprising open reading frames which encode Human enterovirus Cstructural proteins or proteases may be obtained by PCR amplificationusing suitably designed primers complementary to nucleic acid sequencesof human Human enterovirus C. Suitable primers may be designed accordingto standard techniques from publicly available nucleic acid sequences ofHuman enterovirus C, such as those complete genome sequences availablein GenBank and accessible at the National Center for BiotechnologyInformation (NCBI). Accession numbers for the complete genome of theHuman enterovirus C poliovirus type I genome include V01149 and V01150.

The expression cassettes of the invention may comprise nucleic acidswhich encode a Human enterovirus C P1 polypeptide. The P1 polypeptide isprocessed (cleaved) by the 3CD protease translated under the control ofthe IRES of the expression cassette to yield VP1, VP3 and/or VP0polypeptides. Combinations of VP0, VP1 and VP3 may self-associate intovirus-like particles.

The Human enterovirus C polypeptide antigen may comprise a Humanenterovirus C coat/capsid VP2 protein, a product of further processingof VP0, in combination with another poliovirus VP0, VP1, VP3 and/or VP4coat/capsid proteins. The combination of Human enterovirus C coat/capsidproteins may take the form of a virus-like particle (VLP), capsomer,complex and/or aggregate.

A gene encoding a Human enterovirus C coat/capsid VP2 protein may beinserted into a plasmid containing a suitable promoter and expressed ina host cell, the protein isolated and used as the basis of animmunogenic composition for use as a vaccine. Furthermore, the geneencoding human poliovirus VP2 protein may be inserted into a plasmidcontaining a suitable promoter and expressed in a host cell, the proteinisolated, and used as the basis of an immunogenic composition for use asa vaccine.

A gene encoding a Human enterovirus C coat/capsid VP4 protein may beinserted into a plasmid containing a suitable promoter and expressed ina host cell, the protein isolated and used as the basis of animmunogenic composition for use as a vaccine. Furthermore, the geneencoding human poliovirus VP4 protein may be inserted into a plasmidcontaining a suitable promoter and expressed in a host cell, the proteinisolated, and used as the basis of an immunogenic composition for use asa vaccine.

A gene encoding a Human enterovirus C coat/capsid VP0 protein may beinserted into a plasmid containing a suitable promoter and expressed ina host cell, the protein isolated and used as the basis of animmunogenic composition for use as a vaccine. Furthermore, the geneencoding human poliovirus VP0 protein may be inserted into a plasmidcontaining a suitable promoter and expressed in a host cell, the proteinisolated, and used as the basis of an immunogenic composition for use asa vaccine.

The invention encompasses a vaccine comprising one or moreimmunologically active antigens comprising one or more Human enterovirusC VP0, VP1, VP2, VP3, VP4 polypeptides, and immunologically activefragments thereof, which vaccine elicits a protective and/orneutralizing immune response directed against a human Enterovirus.

In an embodiment, the expression cassette consists essentially of anucleic acid encoding a Human enterovirus C P1 polyprotein, an IRES andan enterovirus 3CD protease under the translational control of the IRES,which protease processes the Human enterovirus C P1 polyprotein intostructural capsid proteins.

In another aspect, the invention provides a vaccine comprising Humanenterovirus A antigen(s), derived from a P1 polyprotein including, VP2,VP4 and/or VP0 proteins, and/or biologically or immunologically activefragments thereof. The Human enterovirus A antigens may be derived fromHEV71 and/or Coxsackievirus A16. The HEV71 antigen may be a single humanenterovirus virus coat/capsid protein. In particular, the HEV71 antigenmay be an HEV71 P1 polyprotein, a VP4, VP2 or VP0 polypeptide, or afragment thereof, which elicits an immune response upon administrationto a human.

In an embodiment, the Human enterovirus A antigen may be a combinationof Human enterovirus A coat/capsid proteins, or immunologically activefragments thereof. For example, the Human enterovirus A antigen maycomprise a poliovirus VP2 protein, in combination with anotherpoliovirus coat/capsid proteins selected from VP1, VP3 and/or VP4polypeptides. The combination of a VP2 polypeptide with other polioviruscoat/capsid proteins may take the form of a virus-like particle (VLP),capsomer, complex and/or aggregate. The combination may be in the formof a fusion protein.

More specifically the invention pertains to vaccines comprising a Humanenterovirus A VP2 coat/capsid protein, or immunogenic fragment thereof,as antigen.

The invention in another aspect includes a vaccine comprising Humanenterovirus A virus like particles (VLPs) and/or capsomers comprisingVP1, VP2, VP3 and/or VP4, or VP0 Human enterovirus A proteins.

A recombinant DNA molecule may be obtained whereby open reading frameswhich encode Human enterovirus A structural proteins and/or proteasesmay be amplified by PCR amplification using suitably designed primerscomplementary to nucleic acid sequences of Human enterovirus A. Suitablydesigned primers may be designed according to standard techniques frompublicly available nucleic acid sequences of HEV71. Accession numbersfor the complete genome of HEV71 include DQ341362, AB204852, AF302996and AY465356.

A recombinant DNA molecule may be obtained whereby open reading frameswhich encode Human enterovirus A P1, VP1, VP2, VP3 and/or VP4, or VP0proteins, and immunologically active fragments thereof, and proteases,may be obtained by PCR amplification using suitably designed primerscomplementary to nucleic acid sequences of Human enterovirus A.

In an aspect of the invention, a Human enterovirus A P1 protein isexpressed to form a polyprotein or polypeptide which is subsequentlycleaved by the 3C or 3CD protease into VP0, VP1 and VP3 proteins. VP0proteins may be further cleaved into VP2 and VP4 proteins. Theenterovirus proteins may self-assemble into VLPs, capsomers, complexesand/or aggregates of enterovirus proteins. Further it will beappreciated that the non-structural genes and the protease genes may beincluded in the same DNA recombinant molecule or in different DNArecombinant molecules, and or expressed from different promoters ortranslation elements.

The expression cassettes of the invention may comprise nucleic acidswhich encode a Human enterovirus A P1 polypeptide. The P1 polypeptide isprocessed (cleaved) by the 3CD protease translated under the control ofthe IRES of the expression cassette to yield VP1, VP3 and VP0polypeptides and immunologically active fragments thereof. Combinationsof VP0, VP1 and VP3 polypeptides may self-associate into virus-likeparticles

A gene encoding a Human enterovirus A VP2 protein, or immunologicallyactive fragment thereof, may be inserted in a plasmid containing asuitable promoter and expressed in a host cell. The isolated Humanenterovirus A antigen, for example, an HEV71 VP2 protein, may beisolated and used as the basis of an immunogenic composition for use asa vaccine or for diagnostic use.

A gene encoding a Human enterovirus A VP4 protein, or immunologicallyactive fragment thereof, may be inserted in a plasmid containing asuitable promoter and expressed in a host cell. The isolated VP4 proteinmay be isolated and used as the basis of an immunogenic composition foruse as a vaccine or for diagnostic use.

A gene encoding a Human enterovirus A VP0 protein, or immunologicallyactive fragment thereof, may be inserted in a plasmid containing asuitable promoter and expressed in a host cell. The isolated VP0 proteinmay be isolated and used as the basis of an immunogenic composition foruse as a vaccine or for diagnostic use.

A gene encoding a Human enterovirus A VP0 protein, or immunologicallyactive fragment thereof, may be operably linked to a suitable promoterand inserted into a plasmid, which plasmid exhibits a Human enterovirusA protease linked to a suitable promoter to provide a doubly recombinantplasmid, which doubly recombinant plasmid may ultimately be expressed ina eukaryotic or prokaryotic cell expression system.

The Human enterovirus A genes and nucleic acids comprised in theexpression cassette may be introduced into an appropriate host cell bymeans known to those skilled in the art. The host cells are propagatedand cultured under conditions which allow expression of Humanenterovirus A genes and proteins.

The invention encompasses a vaccine comprising one or moreimmunologically active antigens comprising one or more Human enterovirusA VP0, VP1, VP2, VP3, VP4 polypeptides, and immunologically activefragments thereof, which vaccine elicits a protective and/orneutralizing immune response directed against a human Enterovirus.

The in an embodiment, the expression cassette consists essentially of anucleic acid encoding a Human enterovirus A P1 polyprotein, an IRES andan enterovirus 3CD protease under the translational control of the IRES,which protease processes the Enterovirus P1 polyprotein into enterovirusstructural capsid proteins.

The structural proteins may take the form of VLPs, capsomers, complexesand/or aggregates.

Indeed, the expression of one or more of the Enterovirus proteins asdescribed herein provides antigens which elicit antibodies, whichantibodies are functional and able to neutralize enteroviruses selectedfrom HEV71, Coxsackievirus A16, Human enterovirus C or any otherpicornavirus to high titre.

The expression of one or more of the Enterovirus proteins suggests thatVP2 and/or VP0 polypeptides contain epitopes recognized by neutralizingantisera.

These functional antibodies surprisingly bind more strongly toEnterovirus VP2 and VP0 polypeptides than to VP1 polypeptides, which VP1polypeptide is understood in the art to be the major capsid proteinrequired for the generation of neutralizing antibodies. It is unexpectedthat VP2 polypeptides are, in fact, important for generatingneutralizing antibodies against HEV71 infection.

Thus, it is surprising that an Enterovirus VP2 polypeptide is thedominant epitope, or antigenic determinant, of the capsid proteins forthe generation of neutralizing antibodies against enterovirus infection.HEV71 VP2 polypeptides, either alone, or in combination with other HEV71capsid proteins, for example VP0 polypeptides, is the dominant antigenwhich elicits neutralizing antibodies directed against HEV71.

In an aspect of the invention, prophylactic vaccinations for preventionof enterovirus infection are contemplated which vaccines incorporate VP0and/or VP2 structural proteins of human enteroviruses into animmunogenic composition. The immunogenic composition or vaccine maycomprise VP0 or VP2 structural proteins from Human enterovirus A,including HEV71 and Coxsackievirus A16, or a combination thereof. Theimmunogenic composition may be administered to a subject to elicitneutralizing antibodies directed against human enteroviruses. Theimmunogenic composition may be comprised in a vaccine which isadministered to a subject for the prevention of hand, foot and mouthdisease infection caused by Human enterovirus A, such as from virusesHEV71 and/or Coxsackievirus A16.

In another aspect of the invention, therapeutic vaccinations areprovided to prevent and/or relieve complications of HEV71 and/orCoxsackievirus A16 infection, for example, the neurologic andcardiovascular complications manifesting as syndromes such asmeningitis, encephalitis, acute flaccid paralysis, pulmonary edema andcardiac failure.

In an aspect of the invention, prophylactic vaccinations for preventionof enterovirus infection are contemplated which vaccines incorporate VP0or VP2 structural proteins from Human enterovirus C, for example PV1,PV2, PV3 structural proteins, or combinations thereof, or biologicallyor immunologically active fragments thereof. The immunogenic compositionmay be comprised in a vaccine which is administered to a subject for theprevention of polio caused by Human enterovirus C, including PV1, PV2,and PV3.

Furthermore, the immunogenic composition or vaccine may comprise acombination of antigens derived from both Human enterovirus C and Humanenterovirus A.

Reference may now be made to various embodiments of the invention asillustrated in the attached figures. In these embodiments it should benoted that the Enterovirus VLPs, capsomers, antigens, and aggregates andspecific constructs of DNA recombinant molecules are given by way ofexample.

It may be concluded that Enterovirus VP2 polypeptides are important toachieve neutralizing antibodies. VP2 polypeptides may be sufficient forformulating a vaccine against an infection with picornaviruses, such asHuman enterovirus A, types HEV71 and Coxsackievirus A16; Humanenterovirus C types 1, 2 and 3: and Human enterovirus D type EV68.

In an aspect of the invention, prophylactic vaccinations for preventionof picornavirus infection are contemplated which vaccinationsincorporate at least VP0 and/or VP2 and VP4 structural proteins of thevirus into an immunogenic composition. The immunogenic composition maybe administered to a subject to elicit neutralizing antibodies directedagainst a picornavirus. The immunogenic composition may be comprised ina vaccine which is administered to a subject for the prevention ofpicornavirus infection.

In another aspect of the invention, therapeutic vaccinations areprovided to prevent and/or relieve complications of picornavirusinfection, for example, the neurologic and cardiovascular complicationsmanifesting as syndromes such as meningitis, encephalitis, acute flaccidparalysis, pulmonary edema and cardiac failure.

In a further aspect of the invention there is provided a vaccinecomposition according to the invention for use in medicine.

In yet another aspect, the invention provides a bivalent, or multivalentvaccine comprising enterovirus VP0 and/or VP2 and/or VP4 antigens. Forexample, Human enterovirus A VP0 and/or VP2 and/or VP4 antigens may becombined. Moreover, the aforementioned antigens from different serotypesof Human enterovirus A, such as antigens from Coxsackievirus A16 andHEV71, may be combined in a vaccine, for example, directed against humanfoot-and-mouth disease.

The enterovirus antigens of bivalent or multivalent vaccines may beproduced from the expression cassettes described herein. The enterovirusantigens may be in the form of virus-like particles, capsomers,complexes, and/or aggregates.

In yet another aspect, the invention provides a bivalent, or multivalentvaccine comprising enterovirus antigen(s), and an antigen providingimmunity against one or more of the following pathogens: diphtheria (D);tetanus (T); pertussis (P); Haemophilus influenzae b (Hib); Hepatitis A(HA) Hepatitis B (HB), and Human Enterovirus 71.

In a pediatric vaccine, other compatible antigens may also be included,e.g., antigens known to be effective against meningitis B, meningitis Aand C, and otitis media.

The amount of picornavirus antigen in each vaccine dose is selected asan amount which induces an immunoprotective response without significantadverse side effects in typical vaccinees. Such amount will varydepending on which specific immunogens are employed. An optimal amountfor a particular vaccine can be ascertained by standard studiesinvolving observation of antibody titers and other responses insubjects. A primary vaccination course may include 2 or 3 doses of avaccine, given at intervals optimal for providing an immunoprotectiveresponse.

The invention thus provides a method for preventing picornavirusinfections in humans, which method comprises treating a human subject inneed thereof with an immunologically effective dose of a vaccineaccording to any aspect of the invention as hereinabove described.

As used herein and in the claims, the terms and phrases set out belowhave the meanings which follow.

“Antibody” refers to an immunoglobulin molecule produced by B lymphoidcells with a specific amino acid sequence evoked in humans or otheranimals by an antigen (immunogen). These molecules are characterized byreacting specifically with the antigen.

“Antibody response” or “humoral response” refers to a type of immuneresponse in which antibodies are produced by B lymphoid cells and aresecreted into the blood and/or lymph in response to an antigenicstimulus. In a properly functioning immune response, the antibody bindsspecifically to antigens on the surface of cells (e.g., a pathogen),marking the cell for destruction by phagocytotic cells and/orcomplement-mediated mechanisms.

“Antigen” refers to any substance that, as a result of coming in contactwith appropriate cells, induces a state of sensitivity and/or immuneresponsiveness and that reacts in a demonstrable way with antibodiesand/or immune cells of the sensitized subject in vivo or in vitro.

“Epitope” refers to the simplest form of an antigenic determinant, on acomplex antigen molecule. This is the specific portion of an antigenthat is recognized by an immunoglobulin or T-cell receptor.

“Fusion protein” refers to a protein antigen formed by expression of apolypeptide made by combining two or more gene sequences derived fromdifferent enterovirus structural proteins.

“Immunologically active fragments” or “biologically active fragments”are fragments of Enterovirus structural proteins which elicitneutralizing antibodies directed against enteroviruses. Accordingly, inthe context of the invention, such immunologically active fragments arepresented to the immune system of an organism in order to affect, ormore preferably to induce, a specific immune response and, thereby,vaccinate or prophylactically protect the organism against an infectionwith an Enterovirus.

Neutralizing antibody immune response is where specialized cells of theimmune system recognize the presentation of such heterologous proteins,peptides or epitopes and launch a specific immune response.

In an embodiment, the vaccine antigen according to the invention caninduce a protective immune response. The term “protective immuneresponse” and/or “neutralizing immune response” as used herein isintended to mean that the vaccinated subject may resist or protectitself against an infection with the pathogenic agent against which thevaccination was done.

“Cellular response” or “cellular host response” refers to a type ofimmune response mediated by specific helper and killer T-cells capableof directly eliminating virally infected or cancerous cells.

“Antigen-presenting cell” refers to the accessory cells of antigeninductive events that function primarily by handling and presentingantigen to lymphocytes. The interaction of antigen presenting cells(APC) with antigens is an essential step in immune induction because itenables lymphocytes to encounter and recognize antigenic molecules andto become activated. Exemplary APCs include macrophages,Langerhans-dendritic cells, Follicular dendritic cells, and B cells.

“B-cell” refers to a type of lymphocyte that produces immunoglobulins orantibodies that interact with antigens.

“Cytotoxic T-lymphocyte” is a specialized type of lymphocyte capable ofdestructing foreign cells and host cells infected with the infectiousagents which produce viral antigens.

The language “consisting essentially of” means that in addition to thosecomponents which are mandatory, other components may also be present incompositions, provided that the essential, basic and/or novelcharacteristics of the compositions are not materially affected by theirpresence.

The language “operably linked” means that the components described arein a relationship permitting them to function in their intended manner.Thus, for example, a promoter “operably linked” to a nucleic acid meansthat the promoter and the nucleic acids of a cistron, or more than onecistron, are combined in such a manner that a single cistronic, a singlebicistronic, or a single multicistronic messenger RNA (mRNA) may beproduced. Protein expression of the messenger RNA may be regulatedaccording to transcriptional/translational elements of the nucleic acidsequence. An IRES sequence which is inserted into an expression cassettein an orientation which is upstream (5′) to a cistron means that theIRES sequence and the nucleic acids of the cistron are ligated in such amanner that translation of the cistronic mRNA is regulated under thecontrol of the IRES.

Reference may now be made to various embodiments of the invention asillustrated in the attached figures. In these embodiments it should benoted that the picornavirus VLPs, capsomers, antigens, and aggregatesand specific constructs of DNA recombinant molecules are given by way ofexample.

Example 1. Description of HEV71 VLP Expression Cassettes and Vectors

Expression cassettes may be constructed through means understood in theart.

1.1 HEV71 VLP Expression Cassette [P1+IRES+3CD]

Features:

-   -   Cassette size: 5172 bp    -   prPs: Pox virus strong early/late synthetic promoter, 43 bp    -   P1: P1 protein coding sequence from EV71-SB12736-SAR-03 (GenBank        Accession: DQ341362) with the addition of a stop codon, 2588 bp    -   IRES: Internal Ribosome Binding site, 585 bp    -   3CD: C and D protein coding sequence of P3 from        EV71-SB12736-SAR-03 (GenBank Accession: DQ341362) with the        addition of a ATG start codon and stop codon, 1940 bp    -   Pac I: Rare cutters, enables cassette to be cloned into pSNX01        (MVA del 3 integration vector)

The cassette was cloned into pDONR221 Gateway entry vector (Invitrogen)to produce pSN01.

See FIG. 1 for a diagram of the expression cassette and the pSN01plasmid.

1.2 HEV71 VLP Expression Cassette fP1+IRES+3C1

Features:

-   -   Cassette size: 3773 bp    -   prPS: Pox virus strong early/late synthetic promoter, 43 bp    -   P1: P1 protein coding sequence from EV71-SB12736-SAR-03 (GenBank        Accession: DQ341362) with the addition of a stop codon, 2588 bp    -   IRES: Internal Ribosome Binding site, 585 bp        -   3CD: C protein coding sequence of P3 from            EV71-SB12736-SAR-03 (GenBank Accession: DQ341362) with the            addition of a ATG a start codon and stop codon, 551 bp    -   Pac I: Rare cutters, enables cassette to be cloned into pSNX01        (MVA del 3 integration vector)

The cassette was cloned into pDONR221 Gateway entry vector (Invitrogen)to produce pSN03.

See FIG. 2 for a diagram of the expression cassette and the pSN03plasmid.

1.3 Methods for obtaining Recombinant Baculoviruses containing theInsert HEV71[P1+IRES+3CD] or [P1+1RES+3C]

The source material for HEV71 P1 plus 3CD was pSN01 and for P1 plus 3Cwas pSN03. The aim was to introduce the HEV71-VLP cassette from pSN01and pSN03 (Entry vectors) into the baculovirus expression plasmid pDEST8(Destination vector) by attL/aaR in vitro recombination using LRCLONASE®, following the instructions in the Invitrogen BAC-TO-BAC®manual (2009).

Two recombinase reactions were set up:

1. pSN01 (EV71-P1+3CD)×pDEST8 to produce pSN07;

2. pSN03 (EV71-P1+3C)×pDEST8 to produce pSN08

pSN07 and pSN08 were used to produce recombinant bacmids bacSN07 andbacSN08 by transforming DH10bac as described in the InvitrogenBAC-TO-BAC® manual. The recombinant bacmids were transfected into Sf9cells to rescue recombinant baculoviruses SN07 and SN08.

The recombinant baculoviruses SN07 and SN08 were used to further infectSf9 cells in 6 well plates to evaluate for expression of processedcapsid proteins. A polyclonal rabbit antiserum specific for VP1 was usedto identify VP1 protein in Western blots of lysates and supernatantsfrom recombinant baculovirus infected Sf9 cells.

1.4 Expression of VP1 in the Supernatant of SN07 Infected Sf9 Cells.

Supernatants of infected Sf9 cells were harvested daily from day 3 today 7 post infection and the proteins were resolved on a 12% SDS-PAGEand transferred to nitrocellulose membranes which were then probed withpolyclonal rabbit anti-VP1 antisera (1:4000 dilution) overnight followedby anti-rabbit conjugated with HRP (1:1000 dilution) for 1 hour at roomtemperature. The Western blots were subsequently developed using TMB.The results are shown in FIG. 3. It was observed that the HEV71 VP1 wasprocessed and that expression of the protein was observed in thesupernatant at day 3 and day 4 post infection and that the amount of VP1expression diminished thereafter. Recombinant baculovirus SN07 oninfection of Sf9 cells generates antigens which are found in thesupernatant.

Example 2. Processed VP1 in both the Supernatants and the Lysates

Sf9 cells were infected at a Multiplicity of Infection (MOI) of 10 withdifferent recombinant baculovirus isolates, including SN07, SN08, acontrol baculovirus bacGUS and mock infected. Supernatants and lysateswere harvested on days 3 and 4 post infection and expression of theproteins evaluated by Western blots using rabbit anti-VP1 antisera(1:4000 dilution) to compare yields of proteins produced by SN07 andSN08. As shown in FIG. 4, expression construct SN07 produced morecleaved VP1 than expression construct SN08 both in the supernatant andin the lysate on both days 3 and 4 post infection.

Example 3. VP1 and VP0 is in the Retentate after Ultrafiltration over a100 kD Molecular Weight Cut Off (MWCO) Membrane

Supernatants from SN07 infected Sf9 cells at day 3 post-infection wereclarified and were passed through AMICON® filters (Millipore Corp.) witha 100 kDa MWCO. The retentate was tested for the presence of processedVP1 and VP0. Since the molecular weight of VP1 is approximately 33 kDa,and the molecular weight of VP0 is 36 KDa, these proteins would not beexpected to remain in the retentate unless they were in an oligomericform. As shown in FIG. 5, these antigens remain in the retentate onpassing the supernatants through a 100 kDa MWCO ultrafilter. Thissuggests that the antigens are associated in an oligomeric form. Thus,it may be concluded that VP1 and VP0 are processed and are in anoligomeric association with other capsid proteins.

Example 4. The Retentate, when used to Immunize Mice, Elicits StrongNeutralizing Antibodies against HEV71

Two groups of outbred white mice were immunized with the supernatantconcentrate from Sf9 infected with SN07 as prepared in Example 3. Theimmunization schedules used are shown in the diagram in FIG. 6.

All mice in the immunized group produced antibodies that neutralizedHEV71 as shown in Table 1. The retentate, when used to immunize mice,elicits strong neutralizing antibodies against HEV71.

TABLE 1 Neutralizing antibodies directed against HEV71. mouse ID neutreciprocal titre m1-1 160 m1-2 640 m1-3 >=2560 m1-4 640 m2-1 80 m2-2 640m2-3 640 m2-4 160 control1 <10 control2 <10

Thus, it may be concluded that the oligomeric proteins are able toelicit neutralizing antibodies in mice.

The oligomeric proteins may be utilized in immunogenic compositionsand/or comprised in vaccines for administration and prophylaxis ofenterovirus infection.

Example 5. VP0 is Expressed in the Lysates of SN07 Infected Sf9 Cells

Sf9 cells were infected with SN07 and the lysates were harvested at 72,96 and 120 hours. Immunoblots were probed with rabbit polyclonalantisera directed against EV71 VP0 and it was shown that VP0 isexpressed at 72 hours post infection. As shown in FIG. 7, the VP0 waspartially cleaved to VP2 starting at 96 hours post infection. Thus bothVP0 and VP2 are present in the lysates of SN07 infected cells.

Example 6. Pooled Neutralizing Sera from Mice Immunized with theOligomeric Antigens in the Supernatant of SN07 Infected Sf9 Cells haveHigh Titres against Recombinant VP2 in ELISA

ELISA plates were coated with equal amounts of recombinant VP1 and VP2and used to test pooled neutralizing mouse antisera. FIG. 8 shows thatthe mice which received the supernatant retentate antigen preparationwere able to bind VP2 better than VP1. Thus, it may be concluded thatthe antibodies are functional and able to neutralize HEV71 to hightitre.

Example 7. Pooled Neutralizing Sera from Mice Immunized with OligomericAntigens in the Supernatant of SN07 Infected Sf9 Cells Bind moreStrongly to VP2 and VP0 than to VP1

The Western blots in FIG. 9 show that the neutralizing pooled mouseantisera bound more strongly to VP2 and VP0 than to VP1 even though thesame amount of total protein was added to each well. This suggests thatVP2 and VP0 contain epitopes recognized by neutralizing antisera.

These functional antibodies surprisingly bind more strongly to VP2 andVP0 than to VP1 which is considered to be the major capsid proteinrequired for the generation of neutralizing antibodies. It is thusrelevant to consider that VP2 is in fact important for generatingneutralizing antibodies.

It may be concluded that the presence of VP2 in a vaccine formulation isimportant to achieve neutralizing antibodies. VP2 may be sufficient forformulating a vaccine against HEV71 and other species A, B and Centeroviruses including human Coxsackievirus A16, Echovirus 30, andpoliovirus types 1, 2 and 3.

It will further be appreciated that the invention includes within itsscope a method of generating an immune response directed againstpoliovirus including the step of administering an effective amount of avaccine comprising a poliovirus antigen.

Example 8. Comparison of the Levels of Neutralizing Antibodies

Immunogenic compositions comprising HEV71 VP1 are used to immunize mice.Similarly, immunogenic compositions comprising HEV71 VP2 and/or VP0 areused to immunize mice. The neutralizing antibody levels in the miceimmunized with HEV71 VP2 and/or VP0 are compared with the neutralizingantibody levels in the mice immunized with HEV71 VP1. The neutralizingantibody levels in the mice immunized with HEV71 VP2 and/or VP0 aresignificantly higher than the neutralizing antibody levels in the miceimmunized with HEV71 VP1.

Example 9. Construction of Recombinant Baculovirus Vector for Expressionof the Human enterovirus C P1 Region and the Protease 3CD from a SingleBicistronic Message

This example provides a method that will result in the efficientproduction of VLPs of Human enterovirus C (poliovirus) by reducing theprotease 3CD mediated killing of baculovirus infected cells.

The construction of a recombinant baculovirus vector for the expressionof the P1 region and the protease 3CD from a single bicistronic messageis shown, for example, in FIG. 1. The 3CD protease gene is translated ina cap-independent fashion under control of the EMCV IRES. This systemprovides the leverage to regulate the expression of protease 3CD, i.e.,evaluate the mutant IRES sequences to find the weakest IRES so that alesser amount of protease is produced compared to the P1 proteins.

A bicistronic vector is constructed in which the plasmid contains apolyhedrin promoter upstream of the coding sequence for the P1.Downstream from the cistrons encoding P1 is an Encephalomyocarditisvirus (EMCV) internal ribosome entry site (IRES) sequence (GenBankaccession number AF113968.2; nucleotides1666 to 2251) followed by thecistrons containing the nucleotide sequence encoding the protease 3CD.The source material for the Human enterovirus C P1 with 3CD may beaccessed at the American Type Culture Collection (ATCC) and synthesizedfrom known Human enterovirus C sequences (GenBank available through theNational Center for Biotechnology Information (NCBI)).

Recombinant baculoviruses are generated using the BAC-TO-BAC® systemaccording to the manufacturer's instructions (Invitrogen). Briefly, LRCLONASE® is used to introduce the Human enterovirus C VLP cassette intothe baculovirus expression plasmid pDEST8 (Destination using vector) byattL/aaR in vitro recombination. The LR CLONASE® reaction is carried outat 25° C. for 1 hour followed by incubation with proteinase K. The LRCLONASE® reaction mix is transformed into Library Efficiency DH5αcompetent cells to obtain expression clones. DNA is isolated from theresultant colonies and are confirmed for the presence of the Humanenterovirus C cassette by restriction enzyme analysis. Recombinantbacmids are constructed by introducing the expression cassette, into thebaculovirus genome harbored in DH10bac cells by T7 transpositionrecombinase. The recombinant bacmids are verified by their whitephenotype on LB agar plates supplemented with 50 μg/ml kanamycin, 7μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/mlIPTG. The PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen) is usedto purify high quality bacmid DNA from DH10Bac E. coli. M13 forward, M13reverse and internal primers from the insert are used to confirm theexistence of the Human enterovirus C cassette. EFFECTENE® transfectionreagent (Qiagen) is used to rescue recombinant baculoviruses bytransfecting the DNAs into Sf9 insect cells. Briefly, Sf9 cells areseeded at 2 million per T25 flask and incubated to adhere for 6 hr at28° C. One microgram of recombinant bacmid DNA is resuspended in 150 μlof DNA condensation buffer and 8 μl of enhancer solution is mixed andincubated at room temperature for 5 minutes. Then 25 μl of EFFECTENE®reagent is added into the DNA mix and incubated for 10 minutes at roomtemperature. One ml culture medium is added into the tubes containingthe transfection complexes and transferred into cell culture flasks anduniformly distributed. At day 3, the supernatant is harvested bycentrifugation at 500 g for 5 minutes. Following transfection, a hightiter viral stock is prepared. Once a high viral stock is obtained, itis employed to determine the optimal times for target proteinexpression.

Example 10. Production of VLPs of HEV71, as well as Human enterovirus C,by means of Reducing the Protease 3CD Mediated Killing of BaculovirusInfected Cells

This experiment describes the construction of recombinant baculovirusvector for the expression of the P1 region and the protease 3CD from asingle bicistronic message. The protease gene 3CD is translated in acap-independent fashion under control of the EMCV IRES, shown in FIG.10. This system provides the leverage to regulate the expression ofprotease 3CD, i.e., evaluate the mutant IRES sequences to find weakestIRES so that a lesser amount of the protease is produced compared to theP1 proteins.

A bicistronic vector was constructed in which a plasmid contains apolyhedrin promoter upstream of the coding sequence for the P1.Downstream from the cistrons encoding P1 is an Encephalomyocarditisvirus (EMCV) internal ribosome entry site (IRES) sequence followed bythe cistrons containing nucleotide sequence encoding the protease 3CD,see FIG. 1. The IRES used in Example 1 contains native EMCV IRESsequence as there are altered forms. The native EMCV IRES sequence inFIG. 10 shows the A6 bifurcation loop in the JK segment. Adding onenucleotide, for example an adenine (A7), reduces the expression. Also,in the construct used in Example 1, the 3CD protease is fused withEncephalomyocarditis virus IRES at the amino-terminus. Importantly outframing the EMCV start codon with 3CD protease coding sequence shouldconsiderably reduce the expression of downstream genes, see FIG. 11.Both modifications are incorporated into the EMCV IRES sequence ofpSN01and named pSN01-M1, as shown in FIG. 12, and synthesized by DNA2.0.

Experiments were designed to characterize the VLPs expressed from themutant EMCV IRES of pSN01-M1. Recombinant baculoviruses were generatedusing the BAC-TO-BAC® system according to the manufacturer'sinstructions (Invitrogen). Briefly, LR CLONASE® was used to introducethe HEV71 VLP cassette into the baculovirus expression plasmid pDEST8(Destination using vector) by attL/aaR in vitro recombination. The LRCLONASE® reaction was carried out at 25° C. for 1 hr followed byincubation with proteinase K. The LR CLONASE® reaction mix wastransformed into Library Efficiency DH5α competent cells to obtainexpression clones. DNA was isolated from the resultant colonies andconfirmed for the presence of the HEV71/poliovirus cassette byrestriction enzyme analysis. Recombinant bacmids are constructed byintroducing the expression cassette of pSN07-M1, into the baculovirusgenome harbored in DH10bac cells by T7 transposition recombinase to givebacSN07-M1. The recombinant bacmids are verified by their whitephenotype on LB agar plates supplemented with 50 μg/ml kanamycin, 7μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/mlIPTG. The PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen) was usedto purify high quality bacmid DNA from DH10Bac E. coli. M13 forward, M13reverse and internal primers from the insert were used to confirm theexistence of the HEV71/poliovirus cassette. EFFECTENE® transfectionreagent (Qiagen) was used to rescue recombinant baculoviruses bytransfecting the DNAs into Sf9 insect cells. Briefly, Sf9 cells wereseeded at 2 million per T25 flask and incubated to adhere for 6 hr at28° C. One microgram of recombinant bacmid DNA was resuspended in 150 μlof DNA condensation buffer and 8 μl of enhancer solution is mixed andincubated at room temperature for 5 minutes. Then 25 μl of EFFECTENE®reagent was added into DNA mix and incubated for 10 minutes at roomtemperature. One ml culture medium was added into the tubes containingthe transfection complexes and transferred into cell culture flasks anduniformly distributed. At day 3 the supernatant was harvested bycentrifugation at 500 g for 5 minutes. Following transfection, a hightiter viral stock is prepared. Once a high viral stock is obtained, itis employed to determine the optimal times for target proteinexpression. For the analysis of the protein of interest, Sf9 cells grownin 10% Grace's insect cell medium (Invitrogen) were resuspended in 1%FBS Sf900-II SFM medium (Invitrogen) to get single cells and were seededat a million per ml density in the flasks and incubated for 4 hr at 28°C. Viral stocks were added into the PBS washed cells at an MOI of 10 androcked gently for 1 hr. The infected cells were washed three times withPBS and the cells were grown in Sf900II-SFM for different time points.Cells were lysed with hypotonic douncing buffer/1% TRITON® X-100(TX-100) (1.5 mM MgCl₂, 50 mM KCl, 20 mM HEPES, 1% TX-100) by rockingthe flask for 30 minutes at room temperature and cell lysates wereprepared by collecting the lysed cells from the flask and centrifugingat 4° C. for 30 minutes at 7000 rpm. The components of the cell lysatesand supernatants were analyzed by immunoblotting and ELISA usingspecific antibodies.

Example 11. Efficient Production of VLPs of HEV71, as well as Humanenterovirus C (Poliovirus) by means of Reducing the Protease 3CDMediated Killing of Baculovirus Infected Cells

The construction of a recombinant baculovirus vector for expression ofthe P1 region and the protease 3CD from a single bicistronic message isshown in FIG. 1. The 3CD protease gene is translated in acap-independent fashion under control of the EMCV IRES, shown in FIG.10. This system provides the leverage to regulate the expression of 3CDprotease, i.e., evaluate the mutant IRES sequences to find the weakestIRES so that a lesser amount of protease is produced compared to the P1proteins.

A bicistronic vector was constructed and the plasmid contains apolyhedrin promoter upstream of the coding sequence for the P1.Downstream from the cistrons encoding P1 is an Encephalomyocarditisvirus (EMCV) internal ribosome entry site (IRES) sequence followed bythe cistrons containing a nucleotide sequence encoding the 3CD protease,see FIG. 1. The IRES used in Example 1 contains native EMCV IRESsequence as there are altered forms. The native EMCV IRES sequence hasthe A6 bifurcation loop in the JK segment, indeed by adding onenucleotide (A7) known to reduce the expression, see FIG. 10. The A6bifurcation loop was modified into A7 in the EMCV IRES sequence of pSN01and named pSN01-M2, see FIG. 13, which is synthesized by DNA2.0.

VLPs expressed by the mutant EMCV IRES of pSN01-M2 were characterized.Recombinant baculoviruses were generated using the BAC-TO-BAC® systemaccording to the manufacturer's instructions (Invitrogen). Briefly, LRCLONASE® was used to introduce the HEV71 VLP cassette into thebaculovirus expression plasmid pDEST8 (Destination using vector) byattL/aaR in vitro recombination. The LR CLONASE® reaction was carriedout at 25° C. for 1 hr followed by incubation with proteinase K. The LRCLONASE® reaction mix was transformed into Library Efficiency DH5αcompetent cells to obtain expression clones. DNA was isolated from theresultant colonies and confirmed for the presence of theHEV71/poliovirus cassette by restriction enzyme analysis. Recombinantbacmids were constructed by introducing the expression cassette ofpSN07-M2, into the baculovirus genome harbored in DH10bac cells by T7transposition recombinase to give bacSN07-M2. The recombinant bacmidswere verified by their white phenotype on LB agar plates supplementedwith 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100μg/ml X-gal, and 40 μg/ml IPTG. The PureLink HiPure Plasmid DNA MiniprepKit (Invitrogen) was used to purify high quality bacmid DNA from DH10BacE. coli. M13 forward, M13 reverse and internal primers from the insertwere used to confirm the existence of the HEV71/poliovirus cassette.EFFECTENE® transfection reagent (Qiagen) was used to rescue recombinantbaculoviruses by transfecting the DNAs into Sf9 insect cells. Briefly,Sf9 cells were seeded at 2 million per T25 flask and incubated to adherefor 6 hr at 28° C. One microgram of recombinant bacmid DNA wasresuspended in 150 μl of DNA condensation buffer and 8 μl of enhancersolution is mixed and incubated at room temperature for 5 minutes. Then25 μl of EFFECTENE® reagent was added into the DNA mix and incubated for10 minutes at room temperature. One ml culture medium was added into thetubes containing the transfection complexes and transferred into cellculture flasks and uniformly distributed. At day 3, the supernatant washarvested by centrifugation at 500 g for 5 minutes. Followingtransfection, a high titer viral stock was prepared. Once a high viralstock was obtained, it was employed to determine the optimal times fortarget protein expression. For the analysis of the protein of interest,Sf9 cells grown in 10% Grace's insect cell medium (Invitrogen) wasresuspended in 1% FBS Sf900-II SFM medium (Invitrogen) to get singlecells and were seeded at a million per ml density in the flasks andincubated for 4 hr at 28° C. Viral stocks were added into the PBS washedcells at an MOI of 10 and rocked gently for 1 hr. The infected cellswere washed three times with PBS and cells were grown in Sf900II-SFM fordifferent time points. Cells were lysed with hypotonic douncingbuffer/1% TX-100 (1.5 mM MgCl₂, 50 mM KCl, 20 mM HEPES, 1% TX-100) byrocking the flask for 30 minutes at room temperature and the celllysates were prepared by collecting the lysed cells from the flask andcentrifuging at 4° C. for 30 minutes at 7000 rpm. The components of thecell lysates and supernatants were analyzed by immunoblotting and ELISAusing specific antibodies.

Example 12. Efficient Production of VLPs of HEV71 as well as Humanenterovirus C (Poliovirus) by Means of Reducing the Protease 3CDMediated Killing of Baculovirus Infected Cells

The construction of a recombinant baculovirus vector for expression ofthe P1 region and the protease 3CD from a single bicistronic message isshown in FIG. 1. The 3CD protease gene is translated in acap-independent fashion under control of the EMCV IRES, shown in FIG.10. This system provides the leverage to regulate the expression ofprotease 3CD, i.e., evaluate the mutant IRES sequences to find theweakest IRES so that a lesser amount of protease is produced compared tothe P1 proteins.

A bicistronic vector was constructed in which the plasmid contains apolyhedrin promoter upstream of the coding sequence for the P1.Downstream from the cistrons encoding P1 is an Encephalomyocarditisvirus (EMCV) internal ribosome entry site (IRES) sequence followed bythe cistrons containing a nucleotide sequence encoding the protease 3CD(FIG. 1). The IRES used in Example 1 contains native EMCV IRES sequenceas there are altered forms. In the pSN01 construct the 3CD protease isfused with Encephalomyocarditis virus polyprotein at the amino-terminus.Out framing the EMCV start codon with 3CD protease coding sequenceshould considerably reduce the expression of downstream genes, see FIG.11. This modification was incorporated into EMCV IRES sequence of pSN01and named pSN01-M3, see FIG. 14, and is synthesized by DNA2.0.

The VLPs expressed by the mutant EMCV IRES of pSN01-M3 were analyzed.Recombinant baculoviruses were generated using the BAC-TO-BAC® systemaccording to the manufacturer's instructions (Invitrogen). Briefly, LRCLONASE® was used to introduce the HEV71 VLP cassette into thebaculovirus expression plasmid pDEST8 (Destination using vector), byattL/aaR in vitro recombination. The LR CLONASE® reaction was carriedout at 25° C. for 1 hr followed by incubation with proteinase K. The LRCLONASE® reaction mix was transformed into Library Efficiency DH5αcompetent cells to obtain expression clones. DNA was isolated from theresultant colonies and confirmed for the presence of HEV71/polioviruscassette by restriction enzyme analysis. Recombinant bacmids wereconstructed by introducing the expression cassette of pSN07-M3, into thebaculovirus genome harbored in DH10bac cells by T7 transpositionrecombinase to give bacSN07-M3. The recombinant bacmids were verified bytheir white phenotype on LB agar plates supplemented with 50 μg/mlkanamycin, 7 μg/ml gentamicin, 10 μg/ml tetracycline, 100 μg/ml X-gal,and 40 μg/ml IPTG. The PureLink HiPure Plasmid DNA Miniprep Kit(Invitrogen) was used to purify high quality bacmid DNA from DH10Bac E.coli. M13 forward, M13 reverse and internal primers from the insert wereused to confirm the existence of the HEV71/poliovirus cassette.EFFECTENE® transfection reagent (Qiagen) was used to rescue recombinantbaculoviruses by transfecting the DNAs into Sf9 insect cells. Briefly,Sf9 cells were seeded at 2 million per T25 flask and incubated to adherefor 6 hr at 28° C. One microgram of recombinant bacmid DNA wasresuspended in 150 μl of DNA condensation buffer and 8 μl of enhancersolution is mixed and incubated at room temperature for 5 minutes. Then25 μl of EFFECTENE® reagent was added into the DNA mix and incubated for10 minutes at room temperature. One ml culture medium was added into thetubes containing the transfection complexes and transferred into cellculture flasks and uniformly distributed. After day 3, supernatant washarvested by centrifugation at 500 g for 5 minutes. Followingtransfection, a high titer viral stock is prepared. Once a high viralstock is obtained, it is employed to determine the optimal times fortarget protein expression.

For the analysis of the protein of interest, Sf9 cells grown in 10%Grace's insect cell medium (Invitrogen) were resuspended in 1% FBSSf900-II SFM medium (Invitrogen) to get single cells and were seeded ata million per ml density in the flasks and incubated for 4 hr at 28° C.Viral stocks were added into the PBS washed cells at a MOI of 10 androcked gently for 1 hr. The infected cells were washed three times withPBS and cells were grown in Sf900II-SFM for different time points. Cellswere lysed with hypotonic douncing buffer/1% TX-100 (1.5 mM MgCl₂, 50 mMKCl, 20 mM HEPES, 1% TX-100) by rocking the flask for 30 minutes at roomtemperature and cell lysates were prepared by collecting the lysed cellsfrom the flask and centrifuging at 4° C. for 30 minutes at 7000 rpm. Thecomponents of the cell lysates and supernatants were analyzed byimmunoblotting and ELISA using specific antibodies.

Example 13. Mutant IRES Construct M2 Expresses Higher Levels of VP1 inthe Supernatant

Recombinant baculoviruses expressing HEV71 capsid proteins under thecontrol of the wild type or mutant EMCV IRES's were evaluated withrespect to the level of expression of the HEV71 capsid proteins from theECMV IRES's. Baculovirus produced VLPs which are expressed under thecontrol of the wild type EMCV IRES from SNO7 of Example 1, and the 3mutant IRES's, M1, M2 and M3 from Examples 10, 11, and 12, respectively,were analyzed with respect to the level of baculovirus expression of theHEV71 capsid proteins. A recombinant baculovirus expressing P1 and 3CDunder different promoters (F) and a control recombinant baculovirusexpressing bacGUS (G) were also included in the study. Sf9 cells wereinfected at an MOI of 5 and both lysates and supernatants were harvestedon day 3 as described in Examples 10-12 above. The lysates andsupernatants were probed with an anti-VP1 antibody to detect theexpression of HEV71 capsid proteins.

The immunoblots of FIG. 15 show that when lysates were probed withantibodies to VP1, the mutants M1 and M2 express higher levels of VP1than the mutant M3 or the construct driven by 2 promoters. However, themutant M2 produced more VP1 in the supernatant. These data are shown inthe top panel.

The bottom panel of FIG. 15 shows the blots which were probed with acontrol anti-gp64 antibody which is directed to the coat protein ofbaculovirus. The immunoblot shows that equivalent amounts of baculoviruswere produced in each sample.

Example 14. Cloning, Expression and Purification of Subunit Vaccinesusing a Baculovirus Expression System

The present invention is intended for the generation and use ofrecombinant HEV71 and poliovirus structural proteins which are fused assingle immunogens to elicit a protective immune response in vaccinatedindividuals. The present invention relates generally to preparingrecombinant HEV71 and/or poliovirus fusion protein vaccine compositionscomprising HEV71 and/or poliovirus subunit protein, or an immunogenicfragment thereof, and an adjuvant in combination with the recombinantHEV71 and/or poliovirus subunit fusion protein. HEV71 and poliovirussubunit fusion proteins may comprise capsid proteins selected from VP1,VP2, VP3, and VP4, combinations thereof, and combinations of immunogenicfragments thereof. In one aspect of this embodiment, the recombinantHEV71 and/or poliovirus fusion protein comprises HEV71 or poliovirussubunit protein and a fusion partner protein in genetic association withthe HEV71 or poliovirus subunit protein. The present inventioncontemplates methods to generate the constructs to express the followingsubunit vaccines in E. coli as well as baculovirus: VP0, VP4-VP2-VP3fusion, VP2-VP3-VP1 fusion.

To generate cDNAs from HEV71 and/or poliovirus encoding VP0, VP4-VP2-VP3fusion, or VP2-VP3-VP1, reverse transcription/polymerase chain reaction(The High Pure Nucleic Acid Kit; Roche) was carried out using purifiedgenomic viral RNA. A forward and a reverse primer was made from the5′and 3′ end of the genes and which primers incorporate a start and stopcodon. The amplified PCR products were digested with EcoRI and NotIrestriction enzymes and cloned into the pFastBac HT vector (Invitrogen)as shown in FIG. 16. The BAC-TO-BAC® expression system from Invitrogenis commercially available and methods were used according to themanufacturer's instructions. The fusion genes are cloned into pFastBacHT donor plasmid and the production of recombinant proteins was basedupon the BAC-TO-BAC® to baculovirus expression system (Invitrogen). ThepFastBac HT donor plasmid carrying the fusion genes was transferred intoa baculovirus shuttle vector (bacmid) by site-specific recombination byT7 transposition recombinase. This was accomplished in E. coli strainDH10Bac. The DH10Bac cells contain the bacmid, which conferred kanamycinresistance and a helper plasmid, which encoded the transposase andconferred resistance to tetracycline. The recombinant pFastBac HTplasmids with the gene of interest were transformed into DH10Bac cellsfor the transposition to generate recombinant bacmids. The transformedcells were serially diluted and each dilution was plated on LB agarplates supplemented with 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10μg/ml tetracycline, 100 μg/ml X-gal, and 40 μg/ml IPTG and incubated forat least 48 hours at 37° C. The white colonies were picked andre-streaked to confirm a white phenotype. Recombinant bacmids wereisolated by the PureLink HiPure Plasmid DNA Miniprep Kit (Invitrogen)and the DNA samples were dissolved in 40 μl of TE (10 mM Tris-HCl pH 8,1 mM EDTA) and used for transfections.

The isolated bacmid DNA was screened for the inserted gene of interestby PCR. EFFECTENE® transfection reagent (Qiagen) was used to rescuerecombinant baculoviruses by transfecting the DNAs into Sf9 insectcells. Briefly, Sf9 cells were seeded at 2 million per T25 flask andincubated to adhere for 6 hr at 28° C. One microgram of recombinantbacmid DNA was resuspended in 150 μl of DNA condensation buffer and 8 μlof enhancer solution is mixed and incubated at room temperature for 5minutes. Then 25 μl of EFFECTENE® reagent was added into DNA mix andincubated for 10 minutes at room temperature. One ml culture medium wasadded into the tubes containing the transfection complexes andtransferred into cell culture flasks and uniformly distributed. At day3, the supernatant was harvested by centrifugation at 500 g for 5minutes. Following transfection, a high titer viral stock is prepared.Once a high viral stock is obtained, it is employed to determine theoptimal times for target protein expression. For the analysis of theprotein of interest, Sf9 cells grown in 10% Grace's insect cell medium(Invitrogen) was resuspended in 1% FBS Sf900-II SFM medium (Invitrogen)to get single cells and are seeded at a million per ml density in theflasks and incubated for 4 hr at 28° C. Viral stocks were added into PBSwashed cells at a MOI of 10 and rocked gently for 1 hr. The infectedcells were washed three times with PBS and cells are grown inSf900II-SFM for different time points. Cells were lysed with hypotonicdouncing buffer/1% TX-100 (1.5 mM MgCl₂, 50 mM KCl, 20 mM HEPES, 1%TX-100) by rocking the flask for 30 minutes at room temperature and thecell lysates were prepared by collecting the lysed cells from the flaskand centrifuging at 4° C. for 30 minutes at 7000 rpm. The expression ofthe heterologous protein in the cells was verified by SDS polyacrylamidegel electrophoresis (SDS-PAGE) and Western blots using the His Probe-HRPantibody (Thermo Scientific) as the probe. Once production ofbaculovirus and the expression of protein were confirmed, the virusstock was amplified to produce a concentrated stock of the baculovirusthat carry the gene of interest. The most appropriate concentration ofthe virus to infect insect cells and the optimum time point for theproduction of the desired protein was also established. For purificationunder denaturing conditions, the cells were lysed in a lysis buffercontaining 6 M guanidinium-HCl in 100 mM NaH₂PO₄, 10 mM Tris, 300 mMNaCl, 10 mM imidazole, pH 8.0 (lysis buffer). The suspension wassonicated on ice with 5 pulses of 1 minute per pulse at a power settingof 60 watts, and was mixed at room temperature for 1 hour. The lysatewas centrifuged at 27K g for 30 min to eliminate cell debris. Thesupernatant was loaded on to a HisTrap (GE healthcare life sciences)column pre-equilibrated with lysis buffer. Following loading, the columnwas washed with 20 column volumes of 6 M guanidinium-HCl in 100 mMNaH₂PO₄, 10 mM Tris, 300 mM NaCl, 40 mM Imidazole, pH 8.0 (wash buffer1), followed by washes with 20 column volumes of 8 M urea in 100 mMNaH₂PO₄, 10 mM Tris, 300 mM NaCl, 40 mM imidazole, pH 8.0 (wash buffer2). The bound protein was eluted with a buffer containing 8 M urea, 100mM NaH₂PO₄, 10 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8 (ElutionBuffer). The fractions containing the protein were pooled and dialyzedagainst PBS, overnight at 4° C. TEV protease was used for removal of thehistidine tag following protein purification according to manufacturer'sinstructions.

Example 15. Expression and Purification of Human enterovirus A and Humanenterovirus C (Poliovirus) Subunit Vaccines in E. coli

The Champion™ pET SUMO Expression System (Invitrogen) produces thehighest levels of soluble protein in E. coli. It utilizes a smallubiquitin-related modifier (SUMO) fusion to enhance the solubility ofexpressed fusion proteins. After expression, the 11 kD SUMO moiety canbe cleaved by the highly specific and active SUMO (ULP-1) protease atthe carboxyl terminal, producing a native protein. Also it containsN-terminal 6×His tag for protein detection and purification.

The construction of pET SUMO-VP0, pET SUMO-VP4-VP2-VP3 and pETSUMO-VP2-VP3-VP1 expression vector for antigenic fusion proteins ofHEV71 and poliovirus, as shown in FIG. 17, is as follows. The fragmentsof VP0, VP4-VP2-VP3 fusion and VP2-VP3-VP1 fusion were used as theantigens for HEV71 and poliovirus subunit vaccines. A SUMO motif and the6×Histag were conjugated to the N-terminus of fusions to aid insolubilization of the protein and purification of the protein,respectively. The antigenic fusion proteins were created by a genecloning technology comprising cloning cDNA sequences encoding respectiveproteins into an expression vector to form expression vectors of pETSUMO-VP0, pET SUMO-VP0, pET SUMO-VP4-VP2-VP3 and pET SUMO-VP2-VP3-VP1.The DNA fragments encoding fusion partners were PCR amplified usingspecific primers which consist of a start codon and a stop codon in theforward and reverse primers, respectively. Ligation of the PCR productwas carried out as follows: fresh PCR product, 10× ligation buffer, pETSUMO vector (25 ng/μl) 2 μl, sterile water added to a total volume of 9μl, and 1 μl T4 DNA ligase (4.0 Weiss units) was added and the ligationreaction incubated at 15° C. for overnight then proceeded totransforming One Shot® Mach1™-T1R (Invitrogen) competent cells. Ten (10)colonies were selected and plasmid DNA isolated from them using thePureLink™ HQ Mini Plasmid Purification Kit (Invitrogen). The plasmidswere analyzed by restriction analysis to confirm the presence and thecorrect orientation of the insert. From the recombinants, plasmid DNAwas isolated as earlier and the plasmids were transformed into BL21(DE3) One Shot® cells (Invitrogen). The transformants were grown andinduction of expression with IPTG at several time points was carried outto determine the optimal time of expression. For each time point, 500 μlwas removed from the induced and uninduced cultures and each cell pelletwas resuspended in 80 μl of SDS-PAGE sample buffer. After centrifugingthe boiled samples, 10 μl of each sample was loaded onto an SDS-PAGE geland electrophoresed.

To scale-up the purification of recombinant fusion protein using aHisTrap nickel column (GE Healthcare Life Sciences), the followingprocedure was adapted. An overnight culture (5%) was inoculated into100-300 ml LB plus 50 μg/ml kanamycin and induced after 2 hrs with 1 mMIPTG. After 2 hrs the cells were harvested by centrifuging at 3000 g for10 minutes. The pellet was resuspended in 10% (total volume of theculture) the binding buffer (20 mM sodium phosphate, 0.5M NaCl and 20 mMimidazole at pH7.4). The cells were sonicated with Misonic UltraSonicateLiquid processor for five times for a minute with a minute gap in an icebucket. The sonicated samples were separated into soluble and insolubleform by centrifuging at 4000 rpm for 1 hr at 4° C. The insolublefraction was resuspended with binding buffer containing 6M urea. Boththe soluble and insoluble fractions were centrifuged at 4000 rpm for 1hr at 4° C. then filtered through 0.22 μm filter unit. A HisTrap columnwas equilibrated with the binding buffer and filtered samples wereloaded onto the column. Next, the column was washed with binding bufferwith 40 mM imidazole and the recombinant protein was eluted with bindingbuffer containing 0.5M imidazole (6M urea for insoluble fraction). Allthe collected samples were tested using Coomassie blue staining protocolfor proteins. Pure recombinant protein containing eluted fraction weredialysed using Merck tubing in Tris-HCl buffer. After the dialysisprotein concentrations were estimated using a Bradford reagent. Thenative protein was generated by using SUMO protease to cleave theN-terminal peptide containing the 6×His tag and SUMO according tomanufacturer's instructions.

Example 16. Antibodies from Pooled Neutralizing Sera from Mice Immunizedwith SN07 Retentate Binds to all the Components of EV71VLPs

The coding sequences of VP1, VP2 and VP0 were separately cloned into thepET SUMO vector as described in Example 15. The individual capsidproteins were expressed in E. coli and purified as described in Example15. The purified proteins VP0, VP1, VP2 and VP3 were subjected toWestern blotting and probed with pooled neutralizing sera used inExample 4 at the dilution of 1:1000. Control mice sera used in Example 4is also included in the studies.

Western blotting results in FIG. 18 show that pooled neutralizing serafrom mice immunized with oliogomeric antigens in the supernatant of SN07infected Sf9 cells bind to all of the VLP components, VP0, VP1, VP2 andVP3.

Example 17. Characterization of HEV71 VLPs—Pull-down of HEV71 VLPs fromCulture Supernatants

20 ml of supernatant from cells infected with recombinant baculovirusSN07 was mixed with 10 ml neutralizing monoclonal antibody(EV18/4/D6-1/F1/G9) and left at room temperature for 1 hr. The mixturewas loaded slowly through a 1 ml column of MabSelect SuRe™ (GE HealthCare) recombinant Protein A, 85 um agarose bead size, which waspre-equilibrated with PBS, then washed with 20 mls PBS and then elutedwith 0.5 ml of 0.1 M glycine-HCl, pH3.0. Fractions were neutralized with30 μl of Tris-HCl, pH8.8. Elution fractions were run on SDS-PAGEfollowed by Coomassie blue staining and destaining.

FIG. 19 shows that all of the VLP components (VP0, VP1 and VP3) arevisible in the Coomassie blue stained SDS-PAGE gel.

Example 18. Analysis of Affinity Column (AFC) Purified HEV71VLPs

An affinity column (AFC) was prepared with a neutralizing monoclonalantibody (EV18/4/D6-1/F1/G9), which was developed using a baculovirusSN07 supernatant retentate. The eluted fraction was analyzed by Westernblotting and is shown in FIG. 20. The eluted fraction in Lanes 1 and 2,probed using an anti-VP1 antibody, shows that VP1 is present in the AFCpurified VLPs. The eluted fraction in Lanes 3 and 4, probed using ananti-VP2 antibody, shows that VP2 is also present in the AFC purifiedVLPs. The eluted fraction in Lanes 5 and 6, probed using an anti-VP2monoclonal antibody, shows that VP2 is present in the AFC purified VLPs.

Example 19. Electron Micrograph of AFC Purified HEV71 VLPs

The fraction eluted from the affinity column (AFC) of Example 18 wasevaluated by electron microscopy. An electron micrograph picture of AFCpurified VLPs is shown in FIG. 21. The results of the electronmicroscopy confirm that HEV71 structural proteins are assembled intoVLPs.

Example 20. Mice Protection Studies using Retentate

Antigen used was a 20× concentrated crude retentate of recombinantbaculovirus SN07 prepared as described above. Vaginal plugs indicativeof pregnancy was determined the morning after mating, designated asEmbryonic Day E0.5. Two doses of 100 μL of retentate mixed with IMJECT®(alum) given i.p. to pregnant dams at E3.5 and, on confirmation ofpregnancy by weight measurement, a second dose was given on E17.5.Initial blood sample was collected from each mouse prior to theadministration of the first vaccine dose, samples were collected weeklythereafter until 14 days after viral challenge of pups. Three groups ofBalb/c mice 20 mice were immunized and then challenged (20 mice), 20mice were mock-immunised and challenged (20 mice), 5 mice non-immunised,non-challenged Five-day-old pups were infected with 50 μl of MP-26Mvirus containing 100× HD₅₀ of MP-26M. All infected animals were observedtwice daily for clinical signs of illness until 14 dayspost-inoculation. Abnomal signs included failure to thrive; weight loss;runting; stomach empty of milk; lethargy; head tilt; hunched posture;ruffled fur; dehydration, hypothermia; limb paralysis. Paralysis wasscored according to the following grading system:

0—normal

1—limb weakness but can still move limbs

2—inability to move affected limbs

3—quadriplegia i.e. inability to move limbs

Animals suffering grade 3 paralysis were euthanased by cervicaldislocation under anaesthetic (HD₅₀). Histopathology was performed onsections from a variety of target organs.

The results of the protection study are shown in Table 2 below.

TABLE 2 83% PROTECTION IN PASSIVE PROTECTION NUMBER OF NUMBER OF NUMBEROF TOTAL NUMBER OF MEDIAN MATED PREGNANT PUBS/ NUMBER SURVIVED SURVIVALSURVIVAL VACCINE MICE MICE LITTER OF PUBS PUBS RATE (DAYS) RETENTATE 202 1, 5 6  5* 83% 14 ALUM/PBS 20 2 5, 7 12 0  0% 5 NO VACCINE/ 5 + 5 2 +1 5, 7, 6 18 18  100%  14 NO CHALLENGE

Two litters of mice were born to VLP vaccinated mothers. One mother had5 pups and showed good maternal care. The second mother had one pup thatwas very weak and did not show good maternal care.

It may be concluded that 83% of mice born to immunized mothers wereprotected from virus challenge.

A summary of the histopathological observations is shown in Table 3.

TABLE 3 SUMMARY OF HISTOPATHOLOGICAL OBSERVATIONS SKELETAL SPINALVACCINE AGE DAYS P1 MOUSE SYMPTOMS SPLEEN LIVER HEART MUSCLE CORD BRAINVLP 19 14 V87P4 NO CLINICAL SIGNS N N(1) N 0.5 N N OF INFECTION 19 14V87P5 NO CLINICAL SIGNS N N(1) N 0.5 N N OF INFECTION 19 14 V87P6 NOCLINICAL SIGNS N N(1) N N N N OF INFECTION ALUM/PBS 10 5 DC4 GRADE3PARALYSIS N N(2) N 3 N N DC5 GRADE3 PARALYSIS N N(2) N 3 N N DC6 GRADE3PARALYSIS N N(2) N 03 N N NO 19 NI V116P4 NO CLINICAL SIGNS N  N(0.5) NN N N VACCINE OF INFECTION V116P5 NO CLINICAL SIGNS N  N(0.5) N N N N OFINFECTION (N = no abnormalities detected)

Five mice gave birth to litters and were included in the vaccineprotection study (11% pregnancy rate). The value of the humane endpoint(HD₅₀), as calculated by the method of Reed and Muench, 1938, wasdetermined to be 2.0×10² TCID₅₀ of MP-26M. 83% of mice born to immunizedmothers were protected from challenge.

The histopathology results showed that 3 mice from mothers in theHEV71-VLP immunized group which were then challenged with HEV71 virusshowed no clinical signs of infection.

However, 3 mice from mothers in the mock-immunized (Alum/PBS) and whichwere then challenged with HEV71 virus succumbed to infection. Two micefrom mothers in the non-immunized, non-challenged group showed noclinical signs of infection.

The VLP vaccine protected one litter of infant mice (5/6 pups fromimmunized mothers vs 0/12 pups from mock-immunized mothers) againstlethal challenge with a B3 genotype mouse-adapted strain of HEV71.

Example 21. Human enterovirus C (Poliovirus-PV) VLP Expression

Variations on poliovirus expression cassettes were constructed togenerate poliovirus VLPs. The expression cassettes all comprised apoliovirus P1 polypeptide and differed with respect to the IRES, whichIRES directs the expression of a poliovirus 3CD protease:(PV-P1+HEV71-IRES+PV-3CD); (PV-P1+EMCV-IRES+PV-3CD);(PV-P1+PV-IRES+PV-3CD). Recombinant baculoviruses harboring thepoliovirus VLP expression cassettes were tested. Lysates from thebaculovirus infected cells were harvested on day 3 post infection andexpression of the poliovirus VP3 was evaluated using rabbit anti-PVP3antibodies (1:2000).

Only the PV-IRES-containing construct produced a VP-3 protein of theexpected size. Unlike in the PV-IRES construct, the poliovirus VP3protein is not processed properly when the 3CD protease is under thecontrol of HEV71-IRES or EMCV-RES and, consequently, adversely affectsthe production of poliovirus VLPs. Thus, the poliovirus PV-VLPexpression cassette harboring the PV-IRES is very efficient forpoliovirus VLP production.

Example 22. ELISA to Demonstrate Poliovirus PV-VLP Assembly

To demonstrate poliovirus VLP generation from the PV-VLP expressioncassette, a two sites ELISA was performed using lysates and supernatantsfrom recombinant baculoviruses carrying a PV-VLP expression cassettewherein the PV-3CD protease is under the control of a poliovirus IRES.Sf9 cells were infected with the recombinant baculoviruses carrying aPV-VLP expression cassette including PV-IRES. Lysates and supernatantswere harvested on day 3 post infection. Formation of the poliovirus VLPswas evaluated using a two sites ELISA.

The two sites ELISA procedure was conducted by preparing proteinA-purified rabbit anti-poliovirus VP3 antibodies as capture antibodiesand diluting in coating buffer, 0.05M Carbonate-bicarbonate buffer, pH9.6, to 50 μg/mL. 100 μl/well of the antibodies was dispensed into NUNCimmunoplates and stored at 4° C. overnight. After washing with PBST(0.05%), the immunoplates were blocked with 200 μl/well blocking buffer,1% casein in PBS, at room temperature for 2 hr. Samples of lysates andsupernatants from recombinant baculoviruses carrying a PV-VLP expressioncassette wherein the PV-3CD protease is under the control of apoliovirus IRES were diluted in diluents, 0.2% casein in PBS, dispensedat 100 μl/well, and were incubated at room temperature for 1 hr and thenwashed with PBST (0.05%).

For detection of VLPs, mouse monoclonal antibodies, anti-VP1 (Clone5-D8/1; Dako), were prepared in 0.2% casein in PBS diluents at a 1:250dilution. The diluted detecting monoclonal antibodies were dispensed at100 μl/well and then incubated at room temperature for 1 hr. Anti-mouseHRP conjugated 2° antibodies were prepared in 0.2% casein in PBSdiluents at a 1:1000 dilution. After washing the plate with PBST(0.05%), 100 μl/well of the diluted 2° antibodies were dispensed and theplate incubated at room temperature for 1 hr. The plates were washedwith washing buffer, PBST (0.05%). SureBlue™ TMB 1(KPL) componentmicrowell peroxidase substrate was dispensed at 100 μl/well andincubated at room temperature for 10 min for development. 100 μl/well ofstop solution, 5 mM NaOH, was added to stop the reaction. The absorbanceof each well was read at an OD of 650 nm.

The results of the two sites ELISA shown in FIG. 23 demonstrate that theVP1 and VP3 proteins are in association with each other indicating thatVLPs are indeed formed from the PV-VLP expression cassette.

Example 23. Construction of HEV71 VLP Expression Cassettes with HEV71IRES (P1+HEV71 IRES+3CD)

The schematic structure of a HEV71 VLP cassette with HEV71-IRES is shownin FIG. 24. The expression cassette is similar to the construct shown inExample 1 (pSN01) except that the expression of the 3CD protease isdriven by the HEV71 IRES rather than the EMCV IRES. The HEV71 IRESsequence is found in GenBank, Accession Number DQ341362.1; nucleotides 1to 747. An HEV71 expression cassette containing vector is introducedinto the baculovirus expression plasmid pDEST8 (Destination vector) byattL/aaR in vitro recombination using LR CLONASE®, following theinstructions in the Invitrogen BAC-TO-BAC® manual (2009). Recombinationbetween the entry vector and pDEST8 produces an expression clone.Expression clones give rise to recombinant bacmid by transformingDH10bac as described in the Invitrogen BAC-TO-BAC® manual. Transfectionof the recombinant bacmid into Sf9 cells rescues the recombinantbaculovirus carrying expression cassette which harbors P1, HEV71 IRESand 3CD.

Further infection of Sf9 cells can be used to evaluate expression ofprocessed capsid proteins with rescued recombinant baculoviruses in 6well plates. A polyclonal rabbit antiserum specific for VP1, VP0 and VP3will identify the assembled VLPs by Western blotting of lysates andsupernatants from recombinant baculovirus infected Sf9 cells.

Example 24. Construction of HEV71 VLP Expression Cassettes with PV-IRES(P1+PV-IRES+3CD)

The expression cassette is similar to the expression cassette in Example24 except that the expression of the 3CD protease is driven by apoliovirus IRES (PV-IRES) rather than an HEV71-IRES. The poliovirus IRESsequence is found in GenBank, Accession Number V01150.12; nucleotides 1to 628.

An HEV71 expression cassette containing vector is introduced into thebaculovirus expression plasmid pDEST8 (Destination vector) by attL/aaRin vitro recombination using LR CLONASE®, following the instructions inthe Invitrogen BAC-TO-BAC® manual (2009). Recombination reaction betweenentry vector and pDEST8 is set up to produce an expression clone. Anexpression clone give rises to recombinant bacmid by transformingDH10bac as described in the Invitrogen BAC-TO-BAC® manual. Transfectionof the recombinant bacmid into Sf9 cells rescues the recombinantbaculovirus carrying expression cassette which harbors P1, PV IRES and3CD.

Further infection of Sf9 cells can be used to evaluate for expression ofprocessed capsid proteins with rescued recombinant baculoviruses in 6well plates. A polyclonal rabbit antiserum specific for VP1, VP0 and VP3will identify the assembled VLPs by Western blotting of lysates andsupernatants from recombinant baculovirus infected Sf9 cells.

The invention claimed is:
 1. An expression cassette comprising apromoter operably linked to a nucleic acid encoding a human EnterovirusP1 polypeptide, wherein the nucleic acid encoding the human EnterovirusP1 polypeptide is operably linked to a nucleic acid encoding an InternalRibosome Entry Site (IRES), wherein the nucleic acid encoding the IRESis operably linked to a nucleic acid encoding a human Enterovirus 3CDprotease, wherein the human Enterovirus 3CD protease is under thetranslational control of the IRES.
 2. The expression cassette of claim1, wherein the human Enterovirus is selected from a species or serotypeof Enterovirus A, Enterovirus B, Enterovirus C and Enterovirus D.
 3. Theexpression cassette of claim 2, wherein the human Enterovirus species orserotype is selected from human Enterovirus EV71 and CocksackievirusA-16.
 4. The expression cassette of claim 2, wherein the humanEnterovirus species or serotype is human interovirus C selected fromPV-1, PV-2 and PV-3.
 5. The expression cassette of claim 1, wherein theIRES is derived from Encephalomyocarditis virus (EMCV) or anEnterovirus.
 6. The expression cassette of claim 5, wherein theEnterovirus is human Enterovirus A or human Enterovirus C.
 7. Theexpression cassette of claim 1, wherein the expression cassettecomprises a nucleic acid encoding a human Enterovirus A P1 polypeptide,wherein the nucleic acid encoding the human Enterovirus A P1 polypeptideis operably linked to a nucleic acid encoding an EMCV IRES, wherein thenucleic acid encoding the EMCV IRES is operably linked to a nucleic acidencoding a human Enterovirus A 3CD protease, wherein the humanEnterovirus A 3CD protease is under the translational control of theEMCV IRES.
 8. The expression cassette of claim 1, wherein the expressioncassette comprises a nucleic acid encoding a human Enterovirus A P1polypeptide, wherein the nucleic acid encoding the human Enterovirus AP1 polypeptide is operably linked to a nucleic acid encoding a humanEnterovirus A IRES, wherein the nucleic acid encoding the humanEnterovirus A IRES is operably linked to a nucleic acid encoding a humanEnterovirus A 3CD protease, wherein the human Enterovirus A 3CD proteaseis under the translational control of the human Enterovirus A IRES. 9.The expression cassette of claim 8, wherein the human Enterovirus A IRESis from human Enterovirus A EV71.